JPH0754323B2 - Immunoassays and immunoassay reagents - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for measuring a substance to be measured and an immunoassay reagent. More specifically, a method and an immunoassay in which the substance to be measured in the test sample solution is measured by an assay method using an antibody such as a monoclonal antibody that specifically binds to the substance to be measured and an antibody that recognizes the Fc portion of the antibody. Regarding reagents,
The present invention relates to a method and a measuring reagent that can detect and / or measure the concentration of a substance to be measured in serum or other body fluids associated with various physical diseases.

[Prior Art] Conventionally, as an immunoassay using an antibody that specifically binds to a substance to be measured such as a monoclonal antibody, a monoclonal antibody bound to an insoluble solid, an antigenic substance, and a labeled monoclonal antibody are bound. Methods for measuring the amount of labeling substance in the obtained immune complex are known (for example, JP-A-57-86051, JP-A-57-118159, and JP-A-57-79455).

[Problems to be Solved by the Invention] However, the conventional measurement methods have a problem that sufficient sensitivity cannot be obtained when the affinity between the substance to be measured and the antibody is weak.

[Means for Solving the Problems] In view of the above problems, the present inventors have proposed a measurement method with sufficient sensitivity regardless of the affinity of an antibody that specifically binds to a substance to be measured such as a monoclonal antibody. As a result of earnest studies to achieve the above, the present invention has been achieved.

That is, the present invention provides a substance to be measured, an antibody that specifically binds to the substance to be measured (first antibody), an antibody obtained by cleaving off the Fc portion of the first antibody on an insoluble solid (second antibody), and a first antibody. Immunoassay for a substance to be measured, characterized in that the substance to be measured is measured by measuring the amount of the substance to be labeled in an immune complex obtained by binding a labeled antibody (labeled antibody) that recognizes the Fc portion of the antibody. Method: antigenic substance to be measured, monoclonal antibody (first antibody) that recognizes the antigenic substance to be measured, monoclonal antibody obtained by cleaving off the Fc portion of the first antibody on the insoluble solid (second antibody), and first antibody Of the antigenic substance characterized by measuring the antigenic substance to be measured by measuring the amount of the labeled substance in the immune complex obtained by binding the labeled antibody (third antibody) that recognizes the Fc portion of Immunoassay method: DUT An antibody that specifically binds to (first antibody), an antibody obtained by cleaving off the Fc portion of the first antibody on an insoluble solid (second antibody), and a labeled antibody that recognizes the Fc portion of the first antibody (labeled antibody) An immunoassay reagent characterized by including as an essential component; a monoclonal antibody that recognizes an antigenic substance to be measured (first antibody); a monoclonal antibody obtained by cleaving off the Fc portion of the first antibody on an insoluble solid ( The second antibody) and a labeled antibody (third antibody) that recognizes the Fc portion of the first antibody are essential constituents.

As the antigenic substance or substance to be measured in the present invention, hormones (insulin, HCG-β, growth hormone, TSH, thyroxine, triiodothyronine, gastrin, glucagon, somatostatin, etc.), enzymes (elastase, amylase, protease) , Lipase, ribonuclease, etc.), serum proteins (IgG, IgA, Ig
M, IgE, IgD, glycoprotein, β ₂ -microglobulin, TBG, glycolipid, etc.), tumor-associated antigen (CEA, α-fetoprotein, ferritin, POA, CA19-9, CA125, etc.), DNA binding protein factor, Cytokines (interferon, interleukin 1, interleukin 2, etc.) or various bacteria, viruses, protozoa (fungus, streptococcus, hepatitis virus, herpes virus, AIDS virus, Toxoplasma gondii, malaria parasite, dysentery amoeba etc.), etc. . Of these, hormones and tumor-associated antigens, which require particularly sensitive measurement systems, are preferred.

As the first antibody, a monoclonal antibody or a polyclonal antibody that specifically binds to the substance to be measured can be used. Of these, a monoclonal antibody having excellent specificity is preferable. Monoclonal antibodies can be prepared by using methods described in the literature for myeloma cells [eg, Gie Keller,
Sea Milstein; Nature (G.Kohler, C.Miltei
n; Natur) Vol.256, PP.495 (1975)], which is a cell-derived antibody, and is an antibody produced from antibody-producing cells of the same clone, the antibody is completely the same, It is known that the specificity for the antigenic substance to be measured is also completely the same.

As the second antibody, a method of using a protease (pepsin, papain, trypsin, plasmin, etc.) for the first antibody [eg, Second Biochemistry Experimental Course 5 Immunobiochemistry Research Method P8
9-P101], and then an antibody from which the Fc portion has been removed by gel filtration chromatography or protein A-affinity chromatography [eg, Zokusei Kagaku Kogaku 5 Immunobiochemistry Research Method P100] is used. it can.

Examples of insoluble solids include siliceous inorganic carriers [glass (porous, matte glass, etc.), silica gel, bentonite, etc.], magnetic materials, and organic carriers (plastic, dextran, paper, etc.). Of these,
Preferred are glass (glass beads and glass test tubes) and plastic (plastic tubes and plastic trays), which are simple and stable to which antibodies can be bound, and which are easy to handle.

As a method for binding the second antibody onto the insoluble solid, a glass and the antibody are chemically bound (using a silane coupling agent and optionally a cross-linking agent) or physically adsorbed [eg, US Pat. No. 4,280,992]. Or the method described in Japanese Patent No. 3652761] or a method in which an antibody is physically adsorbed to a plastic [eg, E. Engval, J. Johnson, P. Perlman; Biokim. Buy office. Actor (E. Engvall,
J. Jons-son, P. Parlmann; Biochim. Biopys. Acta), 251.
(1971) 427 to 434].

As the antibody used for the labeled antibody or the third antibody, an antibody that specifically recognizes the Fc portion of the first antibody [eg, an anti-mouse IgG Fc antibody if the first antibody is a mouse IgG monoclonal antibody and the cleavage removal portion is the Fc portion, Alternatively, the first binding protein is a mouse IgA monoclonal antibody and the cleavage-elimination part is Fc.
If it is a part, anti-mouse IgA Fc antibody etc.] can be mentioned.

As the labeling substance for labeling the labeled antibody or the third antibody, enzymes (peroxidase, alkaline phosphatase, β-galactosidase, etc.), radioisotopes ( ³ H, ¹²⁵ I, etc.), fluorescent substances (fluorescein isothiocyanate, rhodamine, etc.) , Chemiluminescent substances (isoluminol derivatives, N-methylacridinium derivatives, etc.)
And so on. Preferred are peroxidase and ¹²⁵ I, which are easily labeled with an antibody and have high sensitivity.

The immune complex according to claim 1, wherein (1) the second antibody on the insoluble solid is bound to the substance to be bound, and then washed, and the substance to be measured is bound to the first antibody, and then washed again. And a method of further binding the first antibody and the labeled antibody, (2)
A method of simultaneously binding a first antibody, a substance to be measured, and a second antibody on an insoluble solid, followed by washing, and further binding the first antibody and a labeled antibody, (3) the first antibody, the substance to be measured,
A method of simultaneously binding the second antibody on the insoluble solid and the labeled antibody, and (4) binding the second antibody on the insoluble solid and the substance to be measured, followed by washing, and the substance to be measured and the first antibody, It can be obtained by a method of simultaneously binding labeled antibodies. For example, (1) the insoluble solid bound with the second antibody and the substance to be measured are incubated in a buffer solution (for example, 5 to 50 ° C. for 5 minutes to 2 days) to be bound, and then the insoluble solid is washed. . Next, the insoluble solid is transferred to a buffer solution containing the first antibody, incubated (for example, 5 to 50 ° C., 5 minutes to 2 days) to be bound, and then the insoluble solid is washed again. Further transfer the insoluble solid to a buffer containing labeled antibody and incubate (eg,
An immune complex is obtained by binding at 5 to 50 ° C. for 5 minutes to 2 days). (2) Incubating the insoluble solid to which the first antibody, the substance to be measured, and the second antibody are bound in a buffer solution (for example, 5 to 50 ° C., 5 minutes to 2 days) to allow binding, and then washing the insoluble solid To do. The insoluble solid is then transferred to a buffer containing labeled antibody and incubated (eg, 5-50
The immunocomplex is obtained by binding at 5 ° C for 5 minutes to 2 days). (3) Immune complex by incubating the first antibody, the substance to be measured, the insoluble solid to which the second antibody is bound, and the labeled antibody in a buffer solution (for example, 5 to 50 ° C., 5 minutes to 2 days) and binding Get the body. Alternatively, (4) the insoluble solid bound with the second antibody and the substance to be measured are incubated in a buffer (for example, 5 to 50 ° C., 5 minutes to 2 minutes).
(Day), and after binding, the insoluble solid is washed. Then transfer the insoluble solid to a buffer containing the first antibody and the labeled antibody,
Incubation (eg 5 to 50 ° C, 5 minutes to 2
Then, an immune complex can be obtained by conjugating them.

The amount of the labeled substance in the immune complex is measured by measuring the enzyme amount by measuring the enzyme activity according to the labeled substance, measuring the radioisotope amount by using a radiation measuring device, and measuring the fluorescent substance amount by a fluorometer. And a method for measuring the amount of chemiluminescent substance by chemiluminescence using an enzyme. For example, when peroxidase is used as the labeling substance, the immune complex is incubated in a chromogenic substrate solution such as ortho-phenylenediamine and hydrogen peroxide solution or 4-aminoantipyrine and hydrogen peroxide solution (for example, 5 to 50 ° C, After 5 minutes to 2 days), the peroxidase activity can be measured by measuring the amount of color development using a spectrophotometer.

In addition to the essential components, the immunoassay reagents according to claims 3 and 4 can include various auxiliary agents for facilitating the use of the immunoassay reagent. When the first antibody, the labeled 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 its enzyme activity, a reaction terminator for terminating the enzymatic reaction, and the like can be included in any combination.

[Examples] Hereinafter, the present invention will be further described 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 was prepared according to the literature [Hillary Koproski, Zenon Stepulski, Kennet Mitchell, Meinhard Herrin; Somatic Cell Jeger. Netix (Hilary Koprowski, Zenon Ste
plewski, Kenneth Mitchell, Meenhard Helyn; Somatic Ce
ll Genetics) Vol. 5, No6, 1979, pp. 957-972].

Briefly, SW1116 cells (available from Dainippon Pharma) 1x
10 ⁶ cells were intravenously administered to BALB / c mice for immunization. One month later, 1 × 10 ⁶ SW1116 cells were administered again, and three days later, the spleen was extracted and splenocytes were collected. After washing with RPM1640 medium, the total amount of splenocytes was 2x10 ⁷ mouse myeloma cells (P3
-NS1 / 1-Ag4.1) and fused at 37 ° C for 1 minute in 1 ml of RPMI1640 medium containing 42.5% polyethylene glycol 1540 and 7.5% dimethylsulfoxide. After 1 minute, the cell suspension was gradually diluted with 5 ml of RPMI1640 medium. After centrifuging and washing the cells, HAT medium (hypoxanthine, aminopterin, thymidine, RPMI1640 medium containing 10% fetal calf serum) was added to 20 ml, and 96
After 0.2 ml each was dispensed into a well microplate and cultured for 2 weeks, the antibody activity of the culture supernatant in the grown well was measured.

Then, cells in wells in which activity was observed were repeatedly cloned using the limiting dilution method. The cloned cells were intraperitoneally administered to mice to grow as ascites tumor, and then ascites was collected. This ascitic fluid monoclonal antibody was purified and isolated using an Affi-Gel Protein A MAPS kit (Bio-Rad) and used for the following studies.

b) Cleavage and removal of Fc portion of anti-CA19-9 monoclonal antibody 20 mg of anti-CA19-9 monoclonal antibody was added to 0.1M NaCl-0.1M.
It was dialyzed against a sodium acetate buffer (pH 4.5). After dialysis,
0.4 mg pepsin (Boehringer Mannheim) was added, and the mixture was incubated at 37 ° C for 6 hours. Next, a 0.1 M sodium hydroxide aqueous solution was added to this solution to adjust the pH to 7, and then gel filtration was performed using a Ultrogel AcA44 (LCB Pharmacia) column (1.5 cm × 90 cm) to remove a fraction containing an Fc portion. Further, undigested monoclonal antibody was removed using an Affi-Gel Protein A MAPS kit to obtain a monoclonal antibody F (ab ') ₂ fraction.

c) Preparation of anti-CA19-9 monoclonal antibody-bound glass beads cleaved and removed from Fc portion According to the method described in US Pat. No. 6,527,61, the surface of glass beads was coated with an anti-CA19-9 monoclonal antibody to which Fc portion was cleaved and removed.

d) Preparation of peroxidase-labeled anti-mouse IgG Fc antibody Anti-mouse IgG Fc antibody (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) 1413
−1424] and binds to peroxidase by the method described in
A peroxidase-labeled anti-mouse IgG Fc antibody was obtained. This reagent is usually 10 to 5000 in a buffer containing 1% bovine serum albumin.
It was used after diluting it twice.

e) Preparation of CA19-9 standard solution SW-1116 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, and the culture supernatant was collected. The concentration of CA19-9 in the culture supernatant was measured using Immunoclon CA19-9 (Fujirebio), and the standard solution was diluted with 1% bovine serum albumin-containing buffer to a concentration of 30,60,120,240 unit / ml. And

f) Preparation of CA19-9 measuring reagent Fc partial cleavage removal anti-CA19-9 monoclonal antibody-bonded glass beads 100, anti-CA19-9 monoclonal antibody 10 μg / m
l containing 0.02M phosphate buffer 12ml, peroxidase-labeled anti-mouse IgG Fc antibody containing 0.02M phosphate buffer 60ml, 1% bovine serum albumin containing 0.02M phosphate buffer 40ml, CA19-9
Standard solution 30, 60, 120, 240 unit / ml 1 ml each, saline 100
0 ml, 60 ml of a substrate solution (ortho-phenylenediamine solution containing hydrogen peroxide), and 350 ml of a 1.5N sulfuric acid aqueous solution were filled in separate containers and then sealed up to prepare a CA19-9 measuring reagent.

g) Measurement of CA19-9 standard solution 50 μl of standard solution containing 240 units / ml of CA19-9, 0.02 M phosphate buffer 100 μ containing 10 μg / ml of anti-CA19-9 monoclonal antibody
l and 1% bovine serum albumin in 0.02M phosphate buffer 3
One Fc partially cleaved and removed anti-CA19-9 monoclonal antibody-bonded glass beads were placed in a test tube containing 00 μl and incubated (37 ° C., 15 minutes), and then the beads were washed with physiological saline. Next, peroxidase-labeled anti-mouse
The beads were transferred into 500 μl of 0.02 M phosphate buffer containing IgG Fc antibody, and incubated (37 ° C., 15 minutes). After washing the beads again with physiological saline, transfer the beads to 500 μl of the substrate solution (hydrogen peroxide-containing ortho-phenylenediamine solution) and incubate (37 ° C, 15 minutes), then add 3 ml of 1.5 N sulfuric acid aqueous solution. In addition, the reaction was stopped. The absorbance of this solution at 492 nm was measured to measure the enzyme activity of the enzyme bound to the beads.

Similarly, the CA19-9 standard solution of 30, 60, 120 unit / ml and 0 blank was also measured.

Fig. 1 shows the measurement results of CA19-9 standard solution by this measurement method.

Comparative Example 1 Measurement of CA19-9 by sandwich method EIA For comparison with Example 1, CA19-9 was measured by a sandwich enzyme immunoassay (EIA) using an anti-CA19-9 monoclonal antibody.

a) Production of anti-CA19-9 monoclonal antibody Same as in Example 1.a).

b) Preparation of anti-CA19-9 monoclonal antibody-bound glass beads According to the method of US Pat. No. 6,527,61, the surface of the glass beads was coated with anti-CA19-9 monoclonal antibody.

c) Preparation of Peroxidase-Labeled Anti-CA19-9 Monoclonal Antibody Anti-CA19-9 monoclonal antibody was prepared according to the literature [S. Yoshitake, M. Imagawa, E. Ishikawa, Etol; Jay. Biochem (S.YOSHITAKE, M.IMAGAWA, E.ISHIKAWA, e
t.al .; J. Biochem., Vol. 92 (1982) 1413-1424] was used to bind to peroxidase to obtain a peroxidase-labeled anti-CA19-9 monoclonal 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 According to Example 1.e).

e) Measurement of CA19-9 standard solution Anti-CA19-9 monoclonal antibody-bound glass beads were placed in a test tube containing 50 μl of standard solution containing 240 unit / ml of CA19-9 and 400 μl of 0.02M phosphate buffer containing 1% bovine serum albumin. After one incubation (37 ° C., 15 minutes), the beads were washed with physiological saline. Next, 0.02 containing anti-CA19-9 monoclonal antibody labeled with peroxidase
The beads were transferred into 500 μl of M phosphate buffer and incubated (37 ° C., 15 minutes). After washing the beads again with physiological saline, the beads were transferred to 500 μl of the substrate solution (hydrogen peroxide-containing ortho-phenylenediamine solution) and incubated (37 ° C., 15 minutes), then 1.5
The reaction was stopped by adding 3 ml of a normal sulfuric acid aqueous solution. 4 of this liquid
The absorbance at 92 nm was measured, and the enzyme activity of the enzyme bound to the beads was measured.

Similarly, the CA19-9 standard solution of 30, 60, 120 unit / ml and 0 blank was also measured.

Fig. 1 shows the measurement results of CA19-9 standard solution by this measurement method.

Example 2 a) Production of anti-HBs monoclonal antibody Monoclonal antibodies against HBs are described in the literature [K. Kunitomo, S. Yahara, H. Saji, H. Saji, Box Sangenis.
T.Hosoi; Vox Sanguinis) Vol.45, No.2,1983, pp.104-11
It was manufactured according to the method described in [1].

Briefly, 500 μg of HBs antigen (Midori Cross) was intraperitoneally administered to BALB / c mice together with Freund's complete adjuvant for immunization. After 10 days, 100 μg of HBs antigen was intravenously administered again, and 3 days later, the spleen was extracted and splenocytes were collected.
After washing with RPMI1640 medium, the total amount of splenocytes was mixed with 2x10 ⁷ mouse myeloma cells (P3-NS1 / 1-Ag 4.1),
The fusion was carried out for 1 minute in 1 ml of RPMI1640 medium containing 42.5% polyethylene glycol 1540 and 7.5% dimethylsulfoxide at 7 ° C. After 1 minute, the cell suspension was added to RPMI1640 medium.
Dilute slowly with 5 ml. After centrifuging and washing the cells, HAT medium (RPMI1640 medium containing hypoxanthine, aminopterin, thymidine, and 10% fetal bovine serum) was used.
Add to 20 ml and add to 96-well microplate.
Each 0.2 ml was dispensed and cultured for 2 weeks, and then the antibody activity of the culture supernatant in the grown well was measured.

Then, cells in wells in which activity was observed were repeatedly cloned using the limiting dilution method. The cloned cells were intraperitoneally administered to mice to grow as ascites tumor, and then ascites was collected. The ascites monoclonal antibody was purified and isolated using an Affi-Gel Protein A MAPS kit (Bio-Rad) and used for the following studies.

b) Anti-H that cleaves and removes the Fc portion of anti-HBs monoclonal antibody
20 mg of Bs monoclonal antibody was dialyzed against 0.1M NaCl-0.1M sodium acetate buffer (pH 4.5). After dialysis, 0.4 mg pepsin (Boehringer Mannheim) was added, and the mixture was mixed at 37 ° C for 6 hours.
Incubated for hours. Next, to this solution was added 0.1 M sodium hydroxide aqueous solution to adjust the pH to 7, and then gel filtration was performed using an Ultrogel AcA44 column (1.5 cm x 90 cm) to obtain Fc.
The fraction containing the part was removed. Further, undigested monoclonal antibody was removed using Affi-Gel Protein A MSPS kit to obtain a monoclonal antibody F (ab ') ₂ fraction.

c) Preparation of Anti-HBs Monoclonal Antibody-Binding Glass Beads with Cleavage of Fc Portion According to the method of US Pat. No. 6,527,61, the surface of glass beads was coated with an anti-HBs monoclonal antibody with Cleavage of Fc segment.

d) Preparation of ¹²⁵ I-labeled anti-mouse IgG Fc antibody An anti-mouse IgG Fc antibody (Binding Site) was used as a reference [Barbara B. Michel, Stanley M. Jigi; Selected Methods in Cellular Immunology (Barbara B. Mishell Stanley M. Shigi; SELECT
ED METHODS IN CELLULAR IMMUNOLOGY) PP.315−316,198
By the method described in 0] combined with ¹²⁵ I, ¹²⁵ I-labeled anti-mouse I
A gG Fc 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 HBs standard solution HBs antigen (40 μg / vial) obtained from Midori-Cross
Diluted with 0.02M phosphate buffer to a concentration of 0.5,1,2.5,5,10n
A HBs standard solution was prepared so as to have g / ml.

f) Preparation of HBs measurement reagent Fc partial cleavage removal 100 anti-HBs monoclonal antibody-bonded glass beads, anti-HBs monoclonal antibody 10 μg / ml included 0.02
12 ml of M phosphate buffer containing ¹²⁵ I-labeled anti-mouse IgG Fc antibody
0.02M phosphate buffer 60ml, containing 1% bovine serum albumin 0.0
2M phosphate buffer 40ml, HBs standard solution 0.5,1,2.5,5,10ng / ml
1 ml of each and 1000 ml of physiological saline were filled in separate containers and then sealed, to prepare a reagent for measuring HBs.

g) Measurement of HBs standard solution 50 μl of standard solution containing 10 ng / ml of HBs, 100 μl of 0.02M phosphate buffer containing 10 μg / ml of anti-HBs monoclonal antibody and 1%
Incubate (37 ° C, 1 ° C, 1 glass beads with anti-HBs monoclonal antibody bound for removal of Fc partial cleavage into a test tube containing 300 μl of 0.02 M phosphate buffer containing bovine serum albumin)
After 5 minutes), the beads were washed with physiological saline.
Next, the beads were transferred to 500 μl of 0.02 M phosphate buffer containing ¹²⁵ I-labeled anti-mouse IgG Fc antibody and incubated (3
7 ° C, 15 minutes). After washing the beads again with physiological saline, the amount of ¹²⁵ I bound to the beads was measured with a γ-ray counter.

Similarly, HBs standard solutions of 0.5, 1, 2.5, 5 ng / ml and 0 blank were also measured.

Fig. 2 shows the measurement results of HBs standard solution by this measurement method.

Comparative Example 2 For comparison with Example 2, HBs antigen was measured by a sandwich radioimmunoassay (RIA) using an anti-HBs monoclonal antibody.

a) Production of anti-HBs monoclonal antibody Same as in Example 2.a).

b) Preparation of anti-HBs monoclonal antibody-bound glass beads According to the method of US Pat. No. 6,527,61, the surface of the glass beads was coated with anti-HBs monoclonal antibody.

c) Preparation of ¹²⁵ I-labeled anti-HBs monoclonal antibody The anti-HBs monoclonal antibody was prepared according to the literature [Barbara B. Michel, Stanley M. Jigi; Selected.
Methodes in Cellular Immunology (Barbara B.
Mishell Stanley M. Shigi; SELECTED METHODS IN CELLUL
AR IMMUNOLOGY) PP.315-316, 1980]
^By binding with ¹²⁵ I, ¹²⁵ I-labeled anti-HBs monoclonal antibody was obtained. This reagent is usually a buffer containing 1% bovine serum albumin
It was used after being diluted 10 to 5000 times.

d) Preparation of HBs standard solution Same as in Example 2.e).

e) Measurement of HBs standard solution One tube of anti-HBs monoclonal antibody-bonded glass beads was placed in a test tube containing 50 μl of standard solution containing 10 ng / ml HBs and 400 μl of 0.02M phosphate buffer containing 1% bovine serum albumin and incubated (37 ° C). For 15 minutes), the beads were washed with physiological saline. Next, the beads were transferred to 500 μl of 0.02 M phosphate buffer containing ¹²⁵ I-labeled anti-HBs monoclonal antibody and incubated (37 ° C., 15 minutes).
After washing the beads again with physiological saline, the amount of ¹²⁵ I bound to the beads was measured with a γ-ray counter.

Similarly, HBs standard solutions of 0.5, 1, 2.5, 5, 10 ng / ml and 0 blank were also measured.

Fig. 2 shows the measurement results of HBs standard solution by this measurement method.

[Effects of the Invention] According to the assay method of the present invention and the immunoassay reagent, highly sensitive assay is possible as compared with the conventional immunoassay method. In particular, according to the measuring method of the present invention as defined in claim 2 and the immunoassay reagent as defined in claim 4, it is possible to carry out highly sensitive measurement as compared with the conventional measuring method using monoclonal antibodies. The conventional measuring method using a monoclonal antibody has a problem that the measuring accuracy is excellent but the measuring sensitivity is low.
According to the measuring method and immunoassay reagent of the present invention, the measuring sensitivity can be improved without lowering the measuring accuracy. Furthermore, when a high-sensitivity measurement method is applied to a test drug, the measurement sensitivity is improved, so that the measurement time can be greatly shortened.

From the above points, the present invention can be applied to the detection of tumor-associated antigens, hormones, viruses, etc., which are contained in trace amounts in serum or other body fluids that require particularly high sensitivity measurement,
In addition, the detection time of these can be shortened.

[Brief description of drawings]

FIG. 1 is a measurement calibration curve of CA19-9 of Example 1 and Comparative Example 1, and FIG. 2 is a measurement calibration curve of HBs antigen of Example 2 and Comparative Example 2.

Claims (4)

[Claims] 1. A substance to be measured, an antibody that specifically binds to the substance to be measured (first antibody), an antibody obtained by cleaving off the Fc portion of the first antibody on an insoluble solid (second antibody), and a first antibody. Fc
By measuring the amount of the labeling substance in the immune complex obtained by binding the labeled antibody recognizing the portion (labeled antibody),
An immunoassay method for a substance to be measured, which comprises measuring the substance to be measured. 2. An antigenic substance to be measured, a monoclonal antibody that recognizes the antigenic substance to be measured (first antibody), a monoclonal antibody obtained by cleaving off the Fc portion of the first antibody on an insoluble solid (second antibody), and An antigen characterized by measuring the antigenic substance to be measured by measuring the amount of a labeling substance in an immune complex obtained by binding a labeled antibody (third antibody) that recognizes the Fc portion of the first antibody Immunoassay for sexual substances. 3. An antibody that specifically binds to a substance to be measured (first antibody), an antibody obtained by cleaving off the Fc portion of the first antibody on an insoluble solid (second antibody), and an Fc portion of the first antibody. An immunoassay reagent comprising a labeled antibody that recognizes (labeled antibody) as an essential component. 4. A monoclonal antibody (first antibody) that recognizes an antigenic substance to be measured, a monoclonal antibody (second antibody) obtained by cleaving off the Fc portion of the first antibody on an insoluble solid, and the Fc portion of the first antibody. An immunoassay reagent comprising a labeled antibody (third antibody) that recognizes as an essential component.