JPS63118656A - Enzyme immunoassay - Google Patents

Abstract

PURPOSE:To obtain satisfactory measurement sensitivity even if a 2nd antibody having a weak antibody titer is used by bringing the immune complex, which is obtd. by bringing the 1st antibody and 2nd antibody simultaneously into reaction, into reaction with an enzyme-labeled antibody which recognizes immune globulin. CONSTITUTION:The antibody (1st antibody) on an insoluble solid which recognizes an antigenic material and the antibody (2nd antibody) which recognizes the antigenic material and the antigenic material derived from an animal different from the 1st antibody are simultaneously brought into reaction to obtain the immune complex. Such immune complex and the enzyme-labeled body (3rd antibody) which recognizes the immune globulin derived from the same animal as the animal of the 2nd antibody are then brought into reaction. The antigenic material is thereafter determined by measuring the amt. of the enzyme. The satisfactory measurement sensitivity is thus obtd. even if the 2nd antibody having the weak antibody titer is used.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to an enzyme immunoassay.

[Prior art 1] Conventionally, as an enzyme immunoassay method, a second antibody labeled with an enzyme is reacted with an antigenic substance bound to a first antibody on an insoluble solid, and the amount of the enzyme is measured (so-called Sand-Deutsch method). ) [For example, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai (eds., "Enzyme Immunoassay" Igakushoin P45 (1982)
].

[Problems to be solved by the invention] However, with this technique, when the antibody titer of the second antibody is low,
Sufficient measurement sensitivity cannot be obtained.

[Means for Solving the Problems] The present inventors have conducted intensive studies on enzyme immunoassay methods that can provide sufficient measurement sensitivity even when using a second antibody with a weak antibody titer, and have developed the present invention. Reached.

That is, the present invention can be obtained by simultaneously reacting a first antibody on an insoluble solid, an antigenic substance recognized by the first antibody, and a second antibody that recognizes the antigenic substance derived from an animal species different from the first antibody. The enzyme is characterized in that the antigenic substance is detected by reacting the immune complex with an enzyme-labeled antibody that recognizes an immunoglobulin derived from the same animal species as the second antibody, and then measuring the amount of the enzyme. It is an immunoassay.

Antigenic substances in the present invention include hormones [insulin, human chorionic gonadotropin β-subunit (
HCG-β), growth hormone, thyroid stimulating hormone (T
SH), thyroxine, triiodothyronine, luteinizing hormone, follicle-stimulating hormone, adrenocorticotropic hormone, parathyroid hormone, etc.), serum proteins (IgG
, IgA, IgM, IgE.

α-fetoprotein, β2-microglobulin,
TBG, etc.), tumor-associated antigens [carcinoembryonic antigen (CEA)]
, ferritin, POA, CA19-9. CA
125 etc.], or bacteria associated with various pathogenic diseases,
virus.

Protozoa [streptococcus, hepatitis virus (hepatitis B virus (t)
LBs), rubella virus, herpes virus, toxoplasma gondii, malaria parasites, and other antigenic components. Preferred are HCG-β, TSH, CEA, and HBs antigens, which require a particularly sensitive measurement system.

Insoluble solids include siliceous inorganic carriers [glass (porous glass, matte glass, etc.), silica gel, bentonite, etc.], magnetic substances, and organic carriers (plastic, dextran, paper, etc.). Among these, those that can be easily and stably bound to antibodies,
Additionally, glass (glass beads and glass test tubes) and plastic (plastic tubes and plastic trays) are easy to handle.

As the first antibody, rabbit was used using a conventionally known method.

Multiclonal antibodies obtained by immunizing mammals such as goats, sheep, and guinea pigs with the above antigenic substances, or cell fusion of antibody-producing cells (derived from mice) against the above antigenic substances and myeloma cells (derived from mice). Examples include mouse-derived monoclonal antibodies obtained by culturing or ascitesing hybridoma cells obtained in this manner.

These antibodies are purified and used by conventionally known purification methods such as ammonium sulfate fractionation, DEAE-cellulose chromatography, and affinity chromatography.

As a method for bonding the first antibody onto an insoluble solid, a method of chemically bonding glass and the antibody (using a silane coupling agent and, if necessary, a crosslinking agent) (for example, Japanese Patent Application Laid-Open No. 108696/1983) or physical adsorption methods (e.g., U.S. Pat. Nos. 4,280,992 and 3,652,761), or methods of physically adsorbing antibodies to plastics [e.g., E. Engvaar, J. J. Johnson, P. Perlman; Biochim, P.O.P., Actor (E, En+; +vall
, J., Johnson, P., Parlmann: B.
iochim, Biophys, Acta), 25
1 (1971) 427-434].

The second antibody may be a multiclonal antibody that recognizes the above-mentioned antigenic substance derived from a different animal species than the first antibody (for example, if the first antibody is rabbit, an antibody from goat, sheep, guinea pig, etc.) or the first antibody is In the case of sheep, antibodies from rabbits, goats, guinea pigs, etc.) If the second or first antibody is a multiclonal antibody that recognizes the above antigenic substance derived from animal species other than mice, antibody-producing cells against the above antigenic substance (antibodies derived from mouse) ) and myeloma cells (derived from mice), and the resulting hybridoma cells are cultured or made into ascites. Forms of secondary antibodies include antiserum, hybridoma culture fluid, It! These include water and immunoglobulins obtained by purifying these.

The immunoglobulin concentration of the second antibody can take various values depending on the concentration of the antigenic substance in the test sample 11 and the concentration of the enzyme-labeled third antibody, but is usually 0.1 to 1000 μg/ml,
Preferably 1 to 200 μg/m! It is.

The immune complex can be obtained by a method in which a first antibody, an antigenic substance, and a second antibody on an insoluble solid are reacted simultaneously. For example, an immune complex can be obtained by simultaneously incubating an insoluble solid bound to a first antibody, a test sample containing an antigenic substance, and a buffer containing a second antibody. Incubation is
Under normal conditions (e.g. 5-50℃, preferably 30-4℃)
0'C temperature, 5 minutes to 2 days, preferably 5 to 30 minutes)
It can be done with

After incubation, a wash solution (eg, distilled water, physiological saline, phosphate buffer, etc.) is added, and the wash solution is subsequently removed by suction using an aspirator.

By performing this operation several times (for example, 2 to 5 times),
Immune complexes can be separated from unreacted substances.

The third antibody is an antibody that recognizes immunoglobulin derived from the same animal species as the second antibody [for example, if the second antibody is a rabbit antibody, anti-rabbit immunoglobulin antibody (guinea pig); if the second antibody is a goat antibody, , anti-goat immunoglobulin antibody (mouse); or when the second antibody is mouse, anti-mouse immunoglobulin antibody (rabbit), etc.).

As for the combination of the first antibody, the second antibody, and the third antibody, it is desirable that the first antibody and the third antibody are derived from the same animal species, since nonspecific adsorption is low. For example, first antibody rabbit, second antibody mouse monoclonal, and third antibody rabbit: first antibody guinea pig, second antibody sheep, and third antibody guinea pig: first antibody sheep, second antibody rabbit, and third antibody Sheep: primary antibody goat,
Examples include second antibody mouse monoclonal, and third antibody goat. Particularly preferred combinations are a first antibody rabbit, a second antibody mouse monoclonal, and a third antibody rabbit; or a first antibody goat, a second antibody mouse monoclone, and and third antibody goat.

Enzymes to label the third antibody include peroxidase,
Examples include alkaline phosphatase and β-galactosidase. Preferably, peroxidase is used because antibody labeling is easy and high sensitivity can be obtained.

Enzymatic labeling of the third antibody can be carried out using conventional methods, such as those described by Niss Yoshitake, M Imagawa, and E.
Ishikawa, Ettor, J. Bahia 1-'7m (S,
YO3HITAKE, ) f, I) IAGAWA,
E, IS old AWA, et, al,, J, Bioc
hem, ) 92 (1982) 1413-142
It can be carried out by the method described in 4.

In the present invention, the reaction between the immune complex obtained by the simultaneous reaction and the enzyme-labeled antibody can be carried out by a conventional method. For example, this is carried out by transferring the immune complex into an enzyme-labeled antibody solution and incubating it. Incubation is carried out under normal conditions (e.g. at a temperature of 5-50'C, preferably 30-40'C, for 5 minutes to 2 days, preferably 5-30'C).
0 minutes).

The reaction product of the immune complex and the enzyme-labeled antibody is washed with a washing solution (e.g. distilled water, physiological saline, phosphate buffer, etc.), and the substrate [e.g. 5-aminosalicylic acid, o-
Phenylenediamine, AB-[2゜2-azinodi(
3-ethylbenzthiazoline)-6'-sulfonic acid]
etc., preferably phenylenediamine] solution, and incubation is performed.

Incubation is carried out under normal conditions (e.g. 5-50
°C, preferably at a temperature of 30 to 40 °C, for 5 minutes to 1 hour,
Preferably, it can be carried out for 5 to 30 minutes).

After incubation, a reaction stop solution (for example, sulfuric acid, hydrochloric acid, etc.) is added, and the enzyme activity is determined by absorbance.

[Examples] Hereinafter, the present invention will be briefly described with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

Example 1 [Measurement of carcinoembryonic antigen (CEA)] (a) Preparation of human CEA standard solution High CEA obtained from colorectal cancer liver metastases by perchloric acid extraction method was subtracted from 10 CEA International 1 No difference standard ( Using Dynabot's CEA measurement kit r CEA-EIA J
5.1 with 0.028 phosphate buffer.
0.30 and 60n! l]/7! It was prepared as follows.

(b) 'N Nari da Danji no Gojio Lyl mice (Balb/c) were immunized with high concentration of human CEA. After 6 weeks, a cell suspension was prepared from the spleen and then about 1 x 108 cells of the spleen and about 2 x 107 mouse myeloma cells were combined with PEG 9!3. It was fused in theory. culturing the fused cells in I-IAT medium;
The target antibody-producing cells (hybridomas) were selected by rough screening. This hybridoma was grown as a single cell group (single clone) by cloning, and then the single clone was inoculated into ascitic fluid in mice. By purifying the obtained ascites, highly specific anti-CEA
Monoclonal immunoglobulins were produced.

(C) Preparation of peroxidase-labeled anti-mouse immunoglobulin antibody Anti-mouse immunoglobulin antibody (manufactured by DakO) was prepared from the above-mentioned document [J, Biochem, 92 (1982) 141
3-1424] to obtain a peroxidase-labeled anti-mouse immunoglobulin antibody'. This reagent was usually diluted 10 to 5000 times with a buffer containing 1% bovine serum albumin.

(d) Preparation of anti-CEA post-clonal antibody-conjugated glass beads According to the method of US Pat. No. 3,652,761, anti-CEA post-clonal (rabbit) antibody (
(manufactured by DakO) was coated.

(e) 1 sample of Σ mice in vitro with 1 piece of anti-CEA cloned (rabbit) antibody-conjugated glass beads and 60 n of human CEA standard solution (1/d,
Add 50μl to anti-CE with 10-100μg of single clone antibody/
300μ of 0.028 phosphate buffer containing m1! After incubation (37° C., 15 minutes) in a microtube, the beads were washed with distilled water. The beads were then transferred into 300 μl of peroxidase-labeled anti-mouse immunoglobulin antibody solution and incubated (37° C. for 15 minutes).

After washing the beads again with distilled water, the beads were incubated in a substrate solution (ortho-phenylenediamine solution containing hydrogen peroxide >400μλ at 37°C).

15 minutes). Next, 13d of 1.5N sulfur was added to stop the reaction. The absorbance of this solution at 492 nm was measured to determine the enzyme activity of the enzyme bound to the beads.

Similarly, human CEA standard solution 30.10. 5 and O
Enzyme activity was measured using nMm, and a calibration curve was created.

FIG. 1 is a calibration curve for human CEA measurement using this measurement method.

Furthermore, the measurement sensitivity, measurement area, and measurement accuracy were as follows.

Measurement sensitivity 1. Ong/Weight measurement head Vj, O~60 nq/m Measurement accuracy 5.0 + 0.4 ng/m (coefficient of variation 8.
0%) 38.2±2.2 ng/ml (coefficient of variation 5.
8%) Comparative Example 1 [Measurement of human CE by conventional Sand-Deutsch method] For comparison with Example 1, human CEA was measured by the conventional Sand-Deutsch method.

(a) Preparation of human eEA standard solution The procedure was as in Example 1(a).

(b) Anti-CEA monoclonal immunization According to Robulin production Example 1(b).

According to Example 1(C), the above anti-CE monoclonal immunoglobulin was conjugated with peroxidase to produce peroxidase-labeled anti-CEA monoclonal immunoglobulin. This reagent is usually prepared in a buffer containing 1% bovine serum albumin.
It was used after being diluted 0 to 5000 times.

((1) Preparation of anti-CEA post-clonal antibody-conjugated glass beads According to Example 1(d).

(e) Quantification of CEA A In a test tube, add 1 piece of anti-CEA clone (rabbit) antibody-conjugated glass beads and human CEA a quasi-solution 60 nQ/ml.
, 50 μ, 1 to 0.02) After incubation in 200 μJ of diluted monophosphate solution (37° C., 215 minutes), the beads were washed with distilled water. Next, peroxidase-labeled anti-CEA monoclonal immunoglobulin solution 300μ
Transfer beads to l and incubate (37°C 11
5 minutes). After washing the beads again with distilled water, the beads were incubated (37° C., 15 minutes) in 400 μl of a substrate solution (ortho-phenylenediamine solution containing hydrogen peroxide). Next, 1.5N 5A acid 3
The reaction was stopped by adding d. The absorbance of this solution at 492 nm was measured to quantify the enzyme activity of the enzyme bound to the beads. Similarly, human CEA standard solution 30.10°5 and On! The enzyme activity was measured using J/ml, and a calibration curve was created.

FIG. 2 is a calibration curve for human CEA measurement according to Comparative Example 1.

Furthermore, the measurement sensitivity, measurement area, and measurement accuracy were as follows.

Measurement sensitivity 4.5 ng/ml Measurement area O~60 ng/W Measurement accuracy 5.3±0.9 ng/Inl1 (coefficient of variation 1
7.0%) 37.5±5.3 ng/ml (coefficient of variation 1
4.1%) Comparative Example 2 [Measurement of human CEA by three-antibody sequential reaction method] For comparison with Example 1, the first antibody and the antigenic substance, the antigenic substance and the second antibody, and the second antibody and the second antibody were tested. Human CEA was measured by sequentially performing three antibody reactions.

(a) Preparation of human CEA standard solution The procedure was as in Example 1(a).

(b) Preparation of anti-CEA clone globulin according to Example 1(b).

(C) Preparation of peroxidase-labeled immunoglobulin antibody according to Example 1 (C).

(d) Preparation of glass beads bound to multiclonal antibodies to anti-CE The procedure of Example 1(d) was followed.

(e) Quantification of CE A In a test tube, 1 piece of anti-CEA clone (rabbit) antibody-conjugated glass beads and human CEA standard solution 60 nMd 50 μ
0.028 liter of phosphate buffer 300. C1f! After incubation (115 minutes at 37°C), the beads were washed with distilled water. Next, the beads were transferred to 300 μl of 0.02 M phosphate buffer containing 10-100 μg/rnll of anti-CEA single antibody and incubated (
After 15 minutes at 37°C, the beads were washed with distilled water. Furthermore, the beads were transferred to 300 μl of peroxidase-labeled anti-mouse immunoglobulin antibody solution and incubated (37° C., 15 minutes). After washing the beads again with distilled water, the beads were added to 400 μl of substrate solution (ortho-phenylenediamine solution containing hydrogen peroxide).
(915 minutes at 37°C).

Next, 3 ml of 1.5N sulfuric acid was added to stop the reaction. The absorbance of this solution at 492 nm was measured, and the enzyme activity of the enzyme bound to the beads was multiplied.

Similarly, human CEA standard solution 30.10.5 and On
The enzyme activity was measured using (]/ml, and a calibration curve was created.

FIG. 3 is a calibration curve for human CEA measurement according to Comparative Example 2.

Furthermore, the measurement sensitivity, measurement area, and measurement accuracy were as follows.

Measurement sensitivity 3.9 n (J/mf! Measurement area 0~60 n (J/d) Measurement accuracy 6.6 ± 0.9 nq/m (coefficient of variation 1
3.6%) 40.2±5.8 nc), 7m1 (coefficient of variation 14.4%) Example 2 [Measurement of thyroid stimulating hormone: 311)] (
a) Preparation of human TSH standard solution Human TSH (5
0μg/vial) to 0.02)! Diluted with phosphate buffer, 50.25. 5. Human TSI+ standard solutions of 2.5 and OIU/Kai were prepared.

(b) Production of anti-TSH monoclonal antibody An anti-human 1511 monoclonal antibody was obtained using the human 1511 described above and according to the method of Example 1(b).

(C) Preparation of anti-TS11 multiclonal antibody-bound glass beads using anti-TSI + multiclonal (rabbit) antibodies (manufactured by Dako) and the method of Example 1(d) to prepare anti-TS11 multiclonal antibody-bound glass beads. Obtained.

(d) Quantification of human TSH In a test tube, 1 piece of anti-TSH double clone (rabbit) antibody-conjugated glass beads and 1 piece of human TSH standard 50 IU/d solution.
00 μl to 10-100 μg of anti-TSH single antibody/
After incubation (37° C., 30 to 60 minutes) in 200 μλ of 0.02) 1 phosphate buffer containing 1 ml, the beads were washed with distilled water. The beads were transferred to 300 μλ of the peroxidase-labeled anti-mouse immunoglobulin antibody solution prepared in Example 1 (C) and incubated (37° C., 30 to 60 minutes). After washing the beads again with distilled water, the beads were added to 400 μl of substrate solution (ortho-phenylenediamine solution containing hydrogen peroxide).
Incubation (10-30℃, 30 minutes)
did.

Next, 3 d of 1,5N sulfuric acid was added to stop the reaction. The absorbance of this solution at 492 nm was measured to quantify the enzyme activity of the enzyme bound to the beads.

Similarly, human TSH standard solution 25. 5. The enzyme activity was measured using 2.5 and OIU/Inl as test samples, and a calibration curve was created.

FIG. 4 is a calibration curve for human TSI (measurement) according to Example 2.

Furthermore, the measurement sensitivity, measurement area, and measurement accuracy were as follows.

Measurement sensitivity 2.0 IU/d Measurement range O ~ 50 Iu/mi Measurement accuracy 2.8 ± 0.2 Iυ/ml (coefficient of variation 7.1%
)26, O±1.4 IU/m1 (coefficient of variation 5.4%)
Comparative Example 3 [Measurement of human TSH by conventional Sand-Inch method] For comparison with Example 2, human TSH was measured by the conventional Sand-Inch method.

(a) Preparation of human SH standard solution Same as Example 2(a).

b) Production Example 2 of anti-TSH monoclonal immunoglobulin
According to (b).

According to Example 1(C), the anti-TSH monoclonal immunoglobulin was combined with peroxidase to produce peroxidase-labeled anti-TSH monoclonal immunoglobulin. This reagent was usually diluted 10 to 5000 times with a buffer containing 1% bovine serum albumin.

(d) Preparation of anti-TSH multiclonal antibody-conjugated glass beads The procedure was as in Example 2 (C).

(e) ``Kiri Nisada'' In a test tube, 1 piece of anti-TSH multiclonal antibody-conjugated glass beads and 100 μl of human TSH Sl + standard 50 II/d solution
Anti-TSH compound antibody 10-100μU/m! After incubation (160 minutes at 37°C) in 200 μλ of a phosphate buffer containing 0.02)i, the beads were washed with distilled water. Next, the beads were transferred to 300 μl of peroxidase-labeled anti-SH monoclonal immunoglobulin solution and incubated (37° C., 60 minutes). After washing the beads again with distilled water, the beads were incubated in 400 μl of a substrate solution (ortho-phenylenediamine solution containing hydrogen peroxide) (37°C,
30 minutes). Next, 13d of 1.5N sulfur was added to stop the reaction. The absorbance of this solution at d92 nm was measured to quantify the enzyme activity of the enzyme bound to the beads.

° Similarly, human TSI + standard solution 25. 5. 2.5
The enzyme activity was measured using OIU/d and a calibration curve was created.

FIG. 5 is a calibration curve for human TSH measurement according to Comparative Example 3.

Furthermore, the measurement sensitivity, measurement area, and measurement accuracy were as follows.

Measurement sensitivity 6.5 IU/d Measurement ffi range O ~ 50 IU/m1 Measurement accuracy 2.6±0.7 IU/ml (coefficient of variation 26.
9%) 25.5±4.3 IU/m! (Coefficient of variation 16.
9%) Comparative Example 4 [Measurement of human TSH by two-antibody sequential reaction method] For comparison with Example 2, human TSH was measured by the conventional three-antibody sequential reaction method.

(a) Preparation of human SH standard solution Same as Example 2(a).

(b) Anti-TS) Single lone antibody S! Construction Example 2 (b
).

(C) Preparation of anti-Ding S11 multiple clone Aramoto-bonded glass beads The procedure was as in Example 2 (C).

(d) Step 11 In a test tube, incubate one anti-TSH double clone (rabbit) antibody-conjugated glass beads and 100 μl of human TSH standard 50 IU/d solution in 200 μl of 0.02) 1-phosphate M buffer. (37°C, 30 minutes), then
The beads were washed with distilled water. Next, the beads were transferred into 300 μl of 0.02M phosphate buffer containing 10-100 μg/mi of anti-TSH single antibody and incubated (
After 30 minutes at 37°C, the beads were washed with distilled water. Furthermore, the beads were transferred into 300 µm of the peroxidase-labeled anti-mouse immunoglobulin antibody solution prepared in Example 1 (C), and incubated (37°C, 30 minutes).

After washing the beads again with distilled water, the beads were incubated in 400 μl of a substrate solution (ortho-phenylenediamine solution containing hydrogen peroxide) (37°C,
30 minutes). Next, 3 d of 1.5N sulfuric acid was added to stop the reaction. The absorbance of this solution at 492 was measured to quantify the enzyme activity of the enzyme bound to the beads.

Similarly, human TSH standard solution 25.5.2.56 and O
Measuring the enzyme activity when ItJ/d is used as a test sample,
A calibration curve was created.

FIG. 6 is a calibration curve for human TSH measurement according to Comparative Example 4.

Furthermore, the measurement sensitivity, measurement area, and measurement accuracy were as follows.

Measurement sensitivity 5.7 IU/d Measurement range 0~501U/d Measurement accuracy 3.0+ 0.6 IU/d (coefficient of variation
20.0%) 28.2±4.711/m1 (coefficient of variation 16.6%) [Effects of the invention] In the conventional Sand-Deutsch method, when the antibody titer of the second antibody to be enzyme-labeled is low, the measurement sensitivity and accuracy are low. Both were so low that normal measurements were impossible. Therefore, we attempted measurement using the two-antibody sequential method, but although a slight increase in sensitivity was observed,
This could not be an effective measurement method.

According to the measurement method of the present invention, sufficient measurement sensitivity can be achieved by 1q even when the antibody titer of the second antibody is low. For example, high sensitivity can be obtained even when the second antibody is an unpurified antibody such as antiserum, culture fluid, or ascites. Furthermore, when a monoclonal antibody is used as the second antibody, highly specific measurement for the antigenic substance of interest becomes possible.

From the above points, the present invention can be particularly applied to diagnostic reagents for CEA 1TSH, HCG-β, and tlBs antigens, which require highly sensitive measurement methods.

[Brief explanation of the drawing]

Figures 1, 2 and 3 are standard curves for human CEA measurement, and Figures 4, 5 and 6 are standard curves for human TSH. Patent Applicant: Sanyo Chemical Industries, Ltd. “Wow Drawing Human CEA Concentrated EC” l/ml) 1 Human-C [A5 Agriculture & (C/J) Human-CEA! ! &('1/r,&) Human-TSH Uno tt<”吃J) Human-T5)1 nok (”z9逗) Hito-Ding SH exchange (calyx 4J) Procedure? 1n Official text (method) % formula % 1. Indication of the case Showa 628f Patent Application No. 83656 2. Name of the invention Enzyme immunoassay method 3. Person making the amendment

Claims (1)

[Scope of Claims] 1. An antibody (first antibody) on an insoluble solid that recognizes an antigenic substance, an antigenic substance, and an antibody (first antibody) that recognizes the antigenic substance derived from an animal species different from the first antibody. After reacting the immune complex obtained by simultaneously reacting two antibodies (two antibodies) with an enzyme-labeled antibody (third antibody) that recognizes immunoglobulin derived from the same animal species as the second antibody, measuring the amount of enzyme. An enzyme immunoassay method characterized in that the antigenic substance is quantified by: 2. The assay method according to claim 1, wherein the second antibody is a monoclonal antibody. 3. Claim 1 in which the enzyme is peroxidase
The measurement method described in Section 2 or Section 2. 4. The antigenic substance is carcinoembryonic antigen (CEA), thyroid stimulating hormone (TSH), human chorionic gonadotropin β-subunit (HCG-β), or hepatitis B virus (
HBs).The measuring method according to any one of claims 1 to 3. 5. The measuring method according to any one of claims 1 to 4, wherein the insoluble solid is glass or plastic. 6. The measuring method according to any one of claims 1 to 5, wherein the first antibody and the third antibody are antibodies derived from the same animal species.