JPH0782019B2 - Immunoassay method and immunoassay reagent - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring an antigenic substance in a test sample solution using monoclonal antibodies of different classes or subclasses, and an immunoassay reagent used in this method, which relates to various physical diseases. Accordingly, the present invention provides a method and a measuring reagent suitable for measuring the concentration of an antigenic substance generated in, for example, serum or other body fluids.

[0002]

2. Description of the Related Art The present inventor has developed an immunoassay using a monoclonal antibody of a different class or subclass from the method described in JP-A-2-205774, that is, by binding an insoluble solid to an antigenic substance to be measured. And a monoclonal antibody that recognizes this antigenic substance (first antibody), a monoclonal antibody that recognizes this antigenic substance and is of a different class or subclass from the first antibody (second antibody), and a labeling substance And a method for measuring an antigenic substance by measuring the amount of the labeled substance in the immune complex obtained by binding the labeled antibody (the third antibody) that recognizes the class or subclass of the second antibody. It has gained. In this method, as the third antibody, specifically, a polyclonal antibody derived from an animal of a different species from the first antibody and the second antibody is used.

[0003]

However, according to the subsequent knowledge obtained by the present inventor, when a polyclonal antibody is used as the third antibody, the specificity of the third antibody is insufficient. It has gradually become clear that sufficient sensitivity may not be obtained due to binding to substances other than the secondary antibody.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that instead of a polyclonal antibody as the third antibody, it is derived from an animal of the same species as the first antibody and the second antibody. It was found that the sensitivity is further improved by using the above monoclonal antibody and the present invention has been reached.

That is, according to the present invention, the antigenic substance to be measured is bound to an insoluble solid and a monoclonal antibody (first antibody) which recognizes this antigenic substance, and which recognizes this antigenic substance, And a monoclonal antibody (second antibody) of a class or subclass different from that of the first antibody
And the amount of the labeled substance in the immune complex obtained by binding the monoclonal antibody (third antibody) that is labeled with the labeled substance and recognizes the class or subclass of the second antibody Immunoassay for an antigenic substance, characterized in that the first antibody, the second antibody and the third antibody are monoclonal antibodies derived from animals of the same species; and binding to insoluble solids And a monoclonal antibody (first antibody) that recognizes an antigenic substance to be measured, and a monoclonal antibody (second antibody) that recognizes this antigenic substance and is of a different class or subclass from the first antibody
And an immunoassay reagent comprising a combination of a labeled monoclonal antibody (third antibody) which is labeled with a labeling substance and recognizes the class or subclass of the second antibody, the first antibody, the second antibody and the third antibody are the same. An immunoassay reagent characterized by being a monoclonal antibody of a species-derived animal.

Examples of the antigenic substance to be measured in the present invention include the antigenic substance to be measured described in JP-A-2-205774. Among these, the method of the present invention is suitable for the measurement of hormones, tumor-associated antigens and viruses, which require particularly sensitive measurement systems.

As the first antibody, a monoclonal antibody that recognizes the antigenic substance to be measured can be used. Monoclonal antibodies can be prepared by the method described in the literature using myeloma cells [eg, G. Kohler, C. Milstein; Natur, Vol. 2].
56, PP. 495 (1975)], the antibody is derived from cells fused by the method described in [56, PP. 495 (1975)] and is produced from antibody-producing cells of the same clone. , The subclasses are exactly the same.

As an insoluble solid, Japanese Patent Laid-Open No. 2057/1990 can be used.
Insoluble solids described in Japanese Patent Publication No. 74 are listed. Among these, preferred ones are capable of easily and stably binding an antibody,
Further, they are glass (glass beads, glass test tubes, etc.) and plastic (plastic tubes, plastic trays, etc.) that are easy to handle.

The method for binding the first antibody to the insoluble solid may be the same as the method described in JP-A-2-205774. That is, a method in which glass and a monoclonal antibody are chemically bound (for example, US Pat. Nos. 4,280,992 and 365,2761) and a method in which an antibody is physically adsorbed on a plastic (for example, e.g.
Engval, J. Johnson, P. Perlman; BioKim. Buy office. Actor (E. Engvall, JJ
ons-son, P. Parlmann; Biochim. Biopys. Acta), 251 (1971) 4
27-434).

As the second antibody, a monoclonal antibody of a class or subclass that recognizes the antigenic substance to be measured and is different from the first antibody can be used. For example, when the first antibody is an IgA class monoclonal antibody, the second antibody can be an IgG class monoclonal antibody, and when the first antibody is an IgG2a subclass monoclonal antibody, the second antibody can be an IgG1 subclass monoclonal antibody.

The third antibody is a monoclonal antibody that specifically recognizes the class or subclass of the second antibody (for example, the first antibody is mouse IgA and the second antibody is mouse I).
If gG, anti-mouse IgG monoclonal antibody, or the first antibody is mouse IgG2a and the second antibody is mouse Ig
For G1, an anti-mouse IgG1 monoclonal antibody or the like) can be used.

As the labeling substance for labeling the third antibody, the labeling substances described in JP-A-2-205774 (enzymes, radioisotopes, fluorescent substances, chemiluminescent substances, etc.) can be used. Of these, preferred are peroxidases that can be easily labeled with an antibody and have high sensitivity.
It is ¹²⁵ I.

As the first antibody, the second antibody and the third antibody, monoclonal antibodies derived from the same species of animal are used, and examples of the same species of animal include mouse, rat and human. Of these, preferred is a mouse capable of producing a monoclonal antibody having a small non-specific binding between antibodies and having a strong affinity with an antigenic substance.

The immune complex is disclosed in Japanese Patent Laid-Open No. 205774/1990.
It can be obtained by the method described in the publication. That is,
(1) The first antibody on the insoluble solid is bound to the antigenic substance to be measured, and then washed, the antigenic substance and the second antibody are bound, and then washed again. A method of binding an antibody and a third antibody, (2) a first antibody on an insoluble solid, an antigenic substance to be measured, and a second antibody are simultaneously bound and washed, and then the second antibody and the second antibody Method of binding three antibodies, (3) method of simultaneously binding first antibody on insoluble solid, antigenic substance to be measured, second antibody, third antibody, and (4) first on insoluble solid It can be obtained by a method in which the antibody is bound to the antigenic substance to be measured, washed, and then the antigenic substance is bound to the second antibody and the third antibody at the same time.

The amount of the labeling substance in the immune complex is determined according to Japanese Patent Laid-Open No.
It can be measured by the method described in JP-A-205774. That is, a method of measuring the amount of enzyme according to the labeling substance by measuring the enzyme activity, a method of measuring the amount of radioisotope using a radiation measuring device, a method of measuring the amount of fluorescent substance with a fluorometer, the amount of chemiluminescent substance Can be performed by a method such as measuring by chemiluminescence using an enzyme.

The immunoassay reagent of the present invention may contain various auxiliary agents for facilitating the use of the immunoassay reagent. For example, when the second antibody or the third antibody is in a solid state, a lysing agent for dissolving them, a washing solution for washing the insolubilized solid in the step of obtaining an immune complex, and a case where the labeling substance is an enzyme A substrate for measuring the enzyme activity, a reaction terminator for stopping the enzymatic reaction, and the like can be included in any combination.

[0017]

EXAMPLES The present invention will be further described below with reference to Examples and Comparative Examples, but the present invention is not limited thereto. Example 1 Measurement of CA19-9 a) Production of anti-CA19-9 monoclonal antibody Monoclonal antibody against CA19-9 is disclosed in Japanese Patent Application Laid-Open No. HEI-2.
-205774, the mouse-derived two clones of IgG1 subclass and IgA class of monoclonal antibodies were obtained and used in the following studies. b) Preparation of Anti-CA19-9 Monoclonal Antibody (IgG1 Subclass) -Binding Glass Beads According to the method described in JP-A-2-205774, IgG1 subclass anti-CA19-9 on the surface of glass beads.
Monoclonal antibody was coated. c) Preparation of anti-mouse IgA monoclonal antibody 100 μg of mouse IgA was intraperitoneally administered to BALB / c mice together with Freund's complete adjuvant for immunization. Two months later, 100 μg of mouse IgA was intravenously administered again, and three days later, the spleen was extracted and splenocytes were collected. After washing with RPMI 1640 medium, the total amount of splenocytes was 2 × 10 ⁷ mouse myeloma cells (P3-NS1 /
1-Ag 4.1) and fused at 37 ° C. for 1 minute in 1 ml of RPMI 1640 medium containing 42.5% polyethylene glycol 1540 and 7.5% dimethylsulfoxide. After 1 minute, the cell suspension was washed with RPMI 16
It was gradually diluted with 5 ml of 40 medium. After centrifugation and washing of the cells, HAT medium (hypoxanthine, aminopterin, thymidine, RP containing 10% fetal bovine serum)
MI 1640 medium) to 20 ml,
0.2 ml each was dispensed to a 96-well microplate and cultured for 2 weeks, and then the antibody activity of the culture supernatant in the grown well was measured. The cells in the wells in which activity was observed were then cloned repeatedly using the limiting dilution method, and Ig
Cells producing anti-mouse IgA monoclonal antibody of G3 subclass were obtained. The cells were cultured in a serum-free culture medium, and the culture supernatant was collected. The monoclonal antibody in this culture solution was purified and isolated using the Affi-Gel Protein A MAPS kit (manufactured by Bio-Rad) and used for the following studies. d) Preparation of peroxidase-labeled anti-mouse IgA monoclonal antibody An anti-mouse IgA monoclonal antibody was prepared according to the literature [S Yoshitake, M. Imagawa, E. Ishikawa, Etol; Jay. Biochem (S.YOSHITAKE, M.IMAGAWA, E.ISHIKAW
A, et.al.; J.Biochem.), Vol.92 (1982) 1413-1424]
By coupling with peroxidase by the method described in 1., a peroxidase-labeled anti-mouse IgA monoclonal antibody was obtained. This reagent was usually diluted with a buffer containing 1% bovine serum albumin 10 to 5000 times before use. e) Preparation of CA19-9 standard solution SW-1116 cells were added to RPMI containing 10% fetal bovine serum.
The cells were cultured in 1640 medium and the culture supernatant was collected. The concentration of CA19-9 in the culture supernatant was adjusted to Immunochron CA19-9.
(Manufactured by Fujirebio Co., Ltd.)
It was diluted with a buffer containing 1% bovine serum albumin to a standard solution of 20,40 units / ml.

F) Preparation of CA19-9 measuring reagent Containing 100 anti-CA19-9 monoclonal antibody (IgG1 subclass) -bound glass beads and 10 μg / ml anti-CA19-9 monoclonal antibody (IgA class).
02M phosphate buffer 12ml, peroxidase-labeled anti-mouse IgA monoclonal antibody-containing 0.02M phosphate buffer 60ml, 1% bovine serum albumin 0.02M
40 ml of phosphate buffer, CA19-9 standard solution 5,1
1 ml each of 0, 20, 40 units / ml, 1000 ml of physiological saline, 60 ml of substrate solution (ortho-phenylenediamine solution containing hydrogen peroxide), and 350 ml of 1.5N sulfuric acid aqueous solution were filled in separate containers, which were then sealed. , C
A19-9 measuring reagent was manufactured. g) Measurement of CA19-9 standard solution Standard solution containing CA19-9 40 unit / ml 5
0 μl, 0.02 M phosphate buffer 10 containing anti-CA19-9 monoclonal antibody (IgA class) 10 μg / ml
An anti-CA19-9 monoclonal antibody (IgG1 subclass) -conjugated glass beads was placed in a test tube containing 0 μl and 300 μl of 0.02 M phosphate buffer containing 1% bovine serum albumin and incubated (37 ° C., 15 minutes).
After that, the beads were washed with physiological saline. Next, the beads were transferred to 500 μl of 0.02 M phosphate buffer containing peroxidase-labeled anti-mouse IgA monoclonal antibody and incubated (37 ° C., 15 minutes). After washing the beads again with physiological saline, the beads were transferred to 500 μl of a substrate solution (hydrogen peroxide-containing ortho-phenylenediamine solution) and incubated (37 ° C.,
After 15 minutes), 3 ml of 1.5 N sulfuric acid aqueous solution 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 measured. Four
The measurement of 0 unit / ml was repeated 10 times, and the average value, standard deviation and CV of the measured values were calculated. Similarly, CA
19-9 standard solution 5, 10, 20 unit / ml and 0
The blank measurement was repeated 10 times, and the average value, standard deviation, and CV of the measured values were calculated. The results are shown in Table 1.

Comparative Example 1 Measurement of CA19-9 by EIA using a goat-derived polyclonal antibody For comparison with Example 1, C19 was determined by an enzyme immunoassay (EIA) using a goat anti-mouse IgA polyclonal antibody.
The measurement of A19-9 was performed. a) Production of anti-CA19-9 monoclonal antibody Example 1. According to a). b) Preparation of anti-CA19-9 monoclonal antibody (IgG1 subclass) -conjugated glass beads Example 1. According to b). c) Preparation of peroxidase-labeled anti-mouse IgA polyclonal antibody Goat anti-mouse IgA polyclonal antibody (manufactured by Kapel) was used as a reference [S. Yoshitake, M. Imagawa, E. Ishikawa, Etol; Jay. Biochem (S.YOSHITAKE, M.IM
AGAWA, E.ISHIKAWA, et.al.; J.Biochem.), Vol.92 (198
2) 1413-1424] to bind to peroxidase to obtain a peroxidase-labeled goat anti-mouse IgA polyclonal antibody. This reagent was usually diluted with a buffer containing 1% bovine serum albumin 10 to 5000 times before use. d) Preparation of CA19-9 standard solution Example 1. According to e). e) Measurement of CA19-9 standard solution CA19-9 standard solution containing 40 units / ml 5
0 μl, anti-CA19-9 monoclonal antibody (IgA class) 10 μg / ml in 0.02M buffer 100 μl
And 1 glass beads conjugated with anti-CA19-9 monoclonal antibody (IgG1 subclass) in a test tube containing 300 μl of 0.02 M phosphate buffer containing 1% bovine serum albumin.
After individual incubation (37, 15 minutes), the beads were washed with physiological saline. Next, the beads were transferred to 500 μl of 0.02 M phosphate buffer containing peroxidase-labeled goat anti-mouse IgA polyclonal antibody,
Incubation (37 ° C, 15 minutes). again,
After washing the beads with physiological saline, the beads are used as a substrate solution (hydrogen peroxide-containing ortho-phenylenediamine solution).
Transfer to 500 μl and incubate (37 ° C, 1
After 5 minutes), 3 ml of 1.5 N sulfuric acid aqueous solution 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 measured. 40
The unit / ml measurement was repeated 10 times, and the average value, standard deviation, and CV of the measured values were calculated. Similarly, CA1
The measurement of the 9-9 standard solution 5, 10, 20 unit / ml and 0 blank was also repeated 10 times, and the average value, standard deviation and CV of the measured values were calculated. The results are shown in Table 1.

[0020]

[Table 1]

Example 2 Measurement of CEA a) Preparation of Anti-CEA Monoclonal Antibody Monoclonal antibody against CEA was disclosed in JP-A 2-20.
It was produced according to the method described in Japanese Patent No. 5774, and two mouse-derived monoclonal antibodies of IgG1 subclass and IgG2a subclass were obtained and used in the following studies. b) Preparation of anti-CEA monoclonal antibody (IgG1 subclass) -bound glass beads According to the method of US Pat. No. 6,527,761, the surface of the glass beads was coated with an IgG1 subclass anti-CEA monoclonal antibody. c) Preparation of anti-mouse IgG2a monoclonal antibody 100 μg of mouse IgG2a together with Freund's complete adjuvant was intraperitoneally administered to BALB / c mice for immunization. Mouse IgG2a 100 μg again after 2 months
Was intravenously administered, and three days later, the spleen was extracted and splenocytes were collected. After washing with RPMI 1640 medium, the total amount of splenocytes was 2 × 10 ⁷ mouse myeloma cells (P3NS1 /
1-Ag 4.1) and fused at 37 ° C. for 1 minute in 1 ml of RPMI 1640 medium containing 42.5% polyethylene glycol 1540 and 7.5% dimethylsulfoxide. After 1 minute, the cell suspension was washed with RPMI 16
It was gradually diluted with 5 ml of 40 medium. After centrifugation and washing of the cells, HAT medium (hypoxanthine, aminopterin, thymidine, RP containing 10% fetal bovine serum)
MI 1640 medium) to 20 ml,
0.2 ml each was dispensed to a 96-well microplate and cultured for 2 weeks, and then the antibody activity of the culture supernatant in the grown well was measured. The cells in the wells in which activity was observed were then cloned repeatedly using the limiting dilution method, and Ig
Cells producing M-class anti-mouse IgG2a monoclonal antibody were obtained. The cells were cultured in a serum-free culture medium, and the culture supernatant was collected. The monoclonal antibody in this culture solution was purified and isolated using the Affi-Gel Protein A MAPS kit (manufactured by Bio-Rad) and used for the following studies. d) Preparation of peroxidase-labeled anti-mouse IgG2a monoclonal antibody An anti-mouse IgG2a monoclonal antibody was used as a reference [S.
Yoshitake, M. Imagawa, E. Ishikawa, Etol; Jay. Biochem (S.YOSHITAKE, M.IMAGAWA, E.IS
HIKAWA, et.al.; J.Biochem.), Vol.92 (1982) 1413-1
424] to bind to peroxidase to obtain a peroxidase-labeled anti-mouse IgG2a monoclonal antibody. This reagent was usually diluted with a buffer containing 1% bovine serum albumin 10 to 5000 times before use. e) Preparation of CEA standard solution High-value CEA obtained by perchloric acid extraction from colorectal cancer liver metastases was measured using WHO's CEA International Reference Standard (63/701), a CEA kit manufactured by Sanyo Kasei Co., Ltd. The concentration is tested by "Glaozyme CEA", and the concentration is 2.5, 5, 10, 20 ng / m
It was diluted with a buffer solution containing 1% bovine serum albumin to give a standard solution.

F) Preparation of CEA measuring reagent 0.02 containing 100 anti-CEA monoclonal antibody (IgG1 subclass) -bound glass beads and 10 μg / ml anti-CEA monoclonal antibody (IgG2a subclass)
M phosphate buffer 12 ml, peroxidase-labeled anti-mouse IgG2a monoclonal antibody-containing 0.02 M phosphate buffer 60 ml, 1% bovine serum albumin-containing 0.02 M
Phosphate buffer 40 ml, CEA standard solution 2.5, 5, 1
0,20 ng / ml 1 ml each, physiological saline 1000 m
1, substrate solution (hydrogen peroxide-containing ortho-phenylenediamine solution) 60 ml, and 1.5N sulfuric acid aqueous solution 350
A CEA measurement reagent was prepared by filling ml in a separate container and then sealing the container. g) Measurement of CEA standard solution 50 μl of standard solution containing CEA 20 ng / ml, anti-C
EA monoclonal antibody (IgG2a subclass) 10
100 μl of 0.02 M phosphate buffer containing μg / ml and 0.02 M phosphate buffer containing 1% bovine serum albumin 30
An anti-CEA monoclonal antibody (IgG1 subclass) -bound glass bead was placed in a test tube containing 0 μl and incubated (37 ° C., 15 minutes), and then the bead was washed with physiological saline. Next, a peroxidase-labeled anti-mouse IgG2a monoclonal antibody-containing 0.0
The beads were transferred into 500 μl of 2M phosphate buffer and incubated (37 ° C., 15 minutes). After washing the beads again with physiological saline, the beads are treated with a substrate solution (hydrogen peroxide-containing ortho-phenylenediamine solution) 500.
After transferring to μl and incubating (37 ° C., 15 minutes), 3 ml of 1.5 N sulfuric acid aqueous solution was added to stop the reaction. The absorbance at 492 nm of this solution was measured,
The enzyme activity of the enzyme bound to the beads was measured. 20 ng
/ Ml measurement was repeated 10 times, the average of the measured values,
The standard deviation and CV were calculated. Similarly, the CEA standard solutions 2.5, 5, 10 ng / ml and 0 blank were measured 10 times each, and the average value, standard deviation, C
V was calculated. The results are shown in Table 2.

Comparative Example 2 Measurement of CEA by EIA using a sheep-derived polyclonal antibody For comparison with Example 2, CEA was measured by an enzyme immunoassay (EIA) using a sheep anti-mouse IgA polyclonal antibody. It was a) Production of anti-CEA monoclonal antibody Example 2. According to a). b) Preparation of anti-CEA monoclonal antibody (IgG1 subclass) -conjugated glass beads Example 2. According to b). c) Preparation of peroxidase-labeled sheep anti-mouse IgG2a polyclonal antibody Sheep anti-mouse IgG2a polyclonal antibody (manufactured by Binding Site) was used as a reference [S. Yoshitake, M. Imagawa, E. Ishikawa,
Etol; Jay. Biochem (S.YOSHITAKE, M.IMAGAW
A, E.ISHIKAWA, et.al.; J.Biochem.), Vol.92 (1982) 1
413-1424] to bind to peroxidase to obtain a peroxidase-labeled sheep anti-mouse IgG2a polyclonal antibody. This reagent was usually diluted with a buffer containing 1% bovine serum albumin 10 to 5000 times before use. d) Preparation of CEA standard solution Example 2. According to e). e) Measurement of CEA standard solution 50 μl of standard solution containing CEA 20 ng / ml, anti-CE
A monoclonal antibody (IgG2a subclass) 10μ
100 μl and 1 of 0.02 M phosphate buffer containing g / ml
0.02M Phosphate Buffer 300 Containing% Bovine Serum Albumin
An anti-CEA monoclonal antibody (I
One glass beads (gG1 subclass) -bonded was placed and incubated (37 ° C., 15 minutes), and then the beads were washed with physiological saline. Next, a peroxidase-labeled sheep anti-mouse IgG2a polyclonal antibody-containing 0.
The beads were transferred into 500 μl of 02M phosphate buffer and incubated (37 ° C., 15 minutes). After washing the beads again with physiological saline, the beads were treated with a substrate solution (hydrogen peroxide-containing ortho-phenylenediamine solution) 5
Transfer to 00 μl and incubate (37 ° C, 15
After that, the reaction was stopped by adding 3 ml of 1.5 N sulfuric acid aqueous solution. The absorbance of this solution at 492 nm was measured, and the enzyme activity of the enzyme bound to the beads was measured. 20
The measurement of ng / ml was repeated 10 times, and the average value, standard deviation and CV of the measured values were calculated. Similarly, the CEA standard solutions 2.5, 5, 10 ng / ml and 0 blank were also measured 10 times each, and the average value, standard deviation, and
CV was calculated. The results are shown in Table 2.

[0024]

[Table 2]

[0025]

EFFECTS OF THE INVENTION According to the measuring method of the present invention, highly sensitive measurement is possible as compared with the conventional measuring method using an antibody derived from a different species than the first antibody and the second antibody as the third antibody. Becomes That is, when an antibody derived from an animal of a species different from that of the first antibody and the second antibody is used as the third antibody, the specificity of the third antibody is insufficient, so that some substances other than the second antibody are used. Binding may occur and sufficient sensitivity may not be obtained, but according to the immunoassay method of the present invention, the first antibody as the third antibody, a monoclonal antibody derived from the same species as the second antibody is used. Therefore, higher specificity can be obtained and the measurement sensitivity can be improved. From the above points, the present invention can be applied to the detection of tumor-associated antigens, hormones, viruses, etc., which are contained in extremely small amounts in serum or other body fluids that require particularly high-sensitivity measurement, and can detect these. The time can be shortened.

Claims (3)

[Claims] 1. A monoclonal antibody (first antibody) which binds to an antigenic substance to be measured and is bound to an insoluble solid, and recognizes this antigenic substance; Monoclonal antibody of a different class or subclass from the antibody (second antibody)
And the amount of the labeled substance in the immune complex obtained by binding the monoclonal antibody (third antibody) that is labeled with the labeled substance and recognizes the class or subclass of the second antibody An immunoassay method for an antigenic substance, which is a method for measuring the amount, wherein the first antibody, the second antibody, and the third antibody are monoclonal antibodies derived from animals of the same species. 2. The immunoassay method according to claim 1, wherein the first antibody, the second antibody and the third antibody are mouse-derived monoclonal antibodies. 3. A monoclonal antibody (first antibody) which binds to an insoluble solid and recognizes an antigenic substance to be measured, a class which recognizes this antigenic substance and is different from the first antibody, or Subclass monoclonal antibody (second antibody)
And an immunoassay reagent comprising a combination of a labeled monoclonal antibody (third antibody) which is labeled with a labeling substance and recognizes the class or subclass of the second antibody, the first antibody, the second antibody and the third antibody are the same. An immunoassay reagent, which is a monoclonal antibody derived from a species of animal.