MEASUREMENT METHOD USING ANTI-IMMUNOCOMPLEX ANTIBODY

Abstract

The invention provides an assay method for an assay system using an anti-immunocomplex antibody, which is able to increase reaction efficiency. The n immunoassay method uses: an anti-hapten rabbit monoclonal antibody immobilized on a water-insoluble carrier, and a labeled anti-immunocomplex antibody,
wherein the method comprises the following steps (i) to (iii): (i) a step of reacting the anti-hapten rabbit monoclonal antibody immobilized on the water-insoluble carrier with a hapten in a solution to be measured, (ii) a step of reacting the labeled anti-immunocomplex antibody with the hapten-anti-hapten rabbit monoclonal antibody immune complex, and (iii) a step of detecting the signal from the label.

Claims

1. An immunoassay method using: an anti-hapten rabbit monoclonal antibody immobilized on a water-insoluble carrier, and a labeled anti-immunocomplex antibody, wherein the method comprises the following steps (i) to (iii): (i) a step of reacting the anti-hapten rabbit monoclonal antibody immobilized on the water-insoluble carrier with a hapten in a solution to be measured, (ii) a step of reacting the labeled anti-immunocomplex antibody with the hapten-anti-hapten rabbit monoclonal antibody immune complex, and (iii) a step of detecting the signal from the label.

2. The method according to claim 1, wherein steps (i), (ii) and (iii) are carried out in that order, and the method has washing steps between steps (i) and (ii) and between steps (ii) and (iii).

3. The method according to claim 1, wherein the anti-immunocomplex antibody is a mouse monoclonal antibody.

4. The method according to claim 1, wherein the label is alkaline phosphatase.

5. The method according to claim 1, wherein the hapten is estradiol, thyroxine or digoxin.

6. The method according to claim 1, wherein step (i) is carried out in the presence of an absorption antibody.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0043] FIG. 1 is a drawing showing an example of a reagent cup to be used for the invention.

[0044] FIG. 2 is a pair of graphs showing the results for the calibration curve of Example 1(3-2).

[0045] FIG. 3 is a pair of graphs showing the results for the calibration curve of Example 2(3-2).

[0046] FIG. 4 is a graph showing the S/N ratio for Example 3 and Comparative Example 1.

[0047] FIG. 5 is a graph showing the results for the calibration curve of Example 4(3-2).

[0048] FIG. 6 is a graph showing the results for the calibration curve of Example 5(4-2).

[0049] FIG. 7 is a graph showing the results for the calibration curve of Example 6(2).

[0050] FIG. 8 is a graph showing the results for the calibration curve of Example 7(4-2).

[0051] FIG. 9 is a graph showing the results for the calibration curve of Example 8(2).

EXAMPLES

Example 1

[0052] An Example of the present invention will now be described in detail, for a method using rabbit monoclonal antibody for estradiol (E2) and anti-immunocomplex mouse monoclonal antibody for the immune complex, with the understanding that the Example is not intended to be restrictive on the invention.

(1) Anti-E2 Rabbit Monoclonal Antibody

[0053] The anti-E2 rabbit monoclonal antibody was obtained by the method described in Japanese Unexamined Patent Publication No. 2009-240300.

(2) Anti-Immunocomplex Mouse Monoclonal Antibody

[0054] Antibody for E2/anti-E2 rabbit monoclonal antibody immune complex (anti-immunocomplex mouse monoclonal antibody) was obtained by the method described in Japanese Patent Publication No. 6031944.

(3) Measurement

[0055] Automatic analysis was carried out by the following method using a fully automatic chemiluminescent enzyme immunoassay device (AIA-CL2400 by Tosoh Corp.).

(3-1) Preparation of Reagent Cup

[0056] A cup having two cells as shown in FIG. 1 was used to prepare a reagent cup for use in automatic analysis. (Hereunder, one cell of the cup will be referred to as the “microparticle cell” and the other as the “conjugate cell”, as per the contents.)

[0057] A solution containing microparticles immobilizing anti-E2 rabbit monoclonal antibody was dispensed into the microparticle cell. A solution containing β-estradiol-6-one-6-(O-carboxymethyloxime) was dispensed into the conjugate cell together with alkaline phosphatase-labeled anti-immunocomplex mouse monoclonal antibody, with reference to PTL 1. The solution was lyophilized and sealed with aluminum to prepare an E2-assay reagent cup.

(3-2) Measurement

[0058] The E2-assay reagent cup prepared in (3-1) was set in the fully automatic chemiluminescent enzyme immunoassay device (AIA-CL2400 by Tosoh Corp.) and measurement was conducted for 6 different samples (cal1 to cal6) of known E2 concentration to construct a calibration curve. For the measurement, 50 μL of a sample of known concentration and 50 μL of a diluent were dispensed into the microparticle cell of the E2-assay reagent cup and reacted for 5 minutes (first-order reaction), after which washing was performed with a cleaning fluid (B/F separation) and the reagent in the conjugate cell that dissolved in the diluent (50 μL) was transferred to the microparticle cell and reacted (secondary reaction, 3 minutes). After completion of the secondary reaction, a second washing was performed with cleaning fluid. Enzyme substrate was then added and the emission intensity was measured. The measurement results are shown in Table 1 and FIG. 2.

[0059] The emission intensity was confirmed to increase in proportion to E2 concentration, making it possible to construct an E2 assay system using anti-immunocomplex antibody.

TABLE-US-00001 TABLE 1 E2 concentration (pg/mL) 0 28.4 101 490 1,090 3,220 Emission 27 2,049 19,110 339,723 1,073,876 2,200,960 intensity (cps)

[0060] These results confirmed that E2 can be detected with high sensitivity in an assay system using anti-hapten rabbit monoclonal antibody as a solid phase antibody and anti-immunocomplex antibody as a labeled antibody.

Example 2

[0061] An Example of the present invention will now be described in detail, for a method using rabbit monoclonal antibody for thyroxine (FT4) and anti-immunocomplex mouse monoclonal antibody for the immune complex, with the understanding that the Example is not intended to be restrictive on the invention.

(1) Anti-FT4 Rabbit Monoclonal Antibody

[0062] The anti-FT4 rabbit monoclonal antibody was obtained by the method described in Japanese Unexamined Patent Publication No. 2009-240300.

(2) Anti-Immunocomplex Mouse Monoclonal Antibody

[0063] The antibody for FT4/anti-FT4 rabbit monoclonal antibody immune complex (anti-immunocomplex mouse monoclonal antibody) was obtained by the method described in Japanese Patent Publication No. 6031944.

(3) Measurement

[0064] Automatic analysis was carried out by the following method using a fully automatic chemiluminescent enzyme immunoassay device (AIA-CL2400 by Tosoh Corp.).

(3-1) Preparation of Reagent Cup

[0065] A cup having two cells as shown in FIG. 1 was used to prepare a reagent cup for use in automatic analysis. A solution containing microparticles immobilizing anti-FT4 rabbit monoclonal antibody was dispensed in an amount of 50 μL into the microparticle cell. A solution containing alkaline phosphatase-labeled anti-immunocomplex mouse monoclonal antibody was dispensed in an amount of 75 μL into the conjugate cell. This was sealed with aluminum to obtain a reagent cup for FT4 assay.

(3-2) Measurement

[0066] The FT4-assay reagent cup prepared in (3-1) was set in the fully automatic chemiluminescent enzyme immunoassay device (AIA-CL2400 by Tosoh Corp.) and measurement was conducted for 6 different samples (cal1 to cal6) of known FT4 concentration to construct a calibration curve. For the measurement, 5 μL of a sample of known concentration and 5 μL of a diluent were dispensed into the microparticle cell of the FT4-assay reagent cup and reacted for 5 minutes (first-order reaction), after which washing was performed with a cleaning fluid (B/F separation) and the reagent in the conjugate cell (50 μL) was transferred to the microparticle cell and reacted (secondary reaction, 3 minutes). After completion of the secondary reaction, a second washing was performed with cleaning fluid. Enzyme substrate was then added and the emission intensity was measured. The measurement results are shown in Table 2 and FIG. 3.

[0067] The emission intensity was confirmed to increase in proportion to FT4 concentration, making it possible to construct an FT4 assay system using anti-immunocomplex antibody.

TABLE-US-00002 TABLE 2 FT4 concentration (ng/dL) 0 0.394 0.967 2.07 4.06 8.7 Emission 3,105 30,926 139,586 323,633 551,883 793,500 intensity (cps)

[0068] These results confirmed that FT4 can be detected with high sensitivity in an assay system using anti-hapten rabbit monoclonal antibody as a solid phase antibody and anti-immunocomplex antibody as a labeled antibody.

Example 3

[0069] An Example of the present invention will now be described in detail, for a method using rabbit monoclonal antibody for estradiol (E2) and anti-immunocomplex mouse monoclonal antibody for the immune complex on a 96-well ELISA plate, with the understanding that the Example is not intended to be restrictive on the invention.

(1) Anti-E2 Rabbit Monoclonal Antibody

[0070] The anti-E2 rabbit monoclonal antibody used was the same antibody as in Example 1.

(2) Anti-Immunocomplex Mouse Monoclonal Antibody

[0071] The anti-immunocomplex mouse monoclonal antibody used was the same antibody as in Example 1.

(3) Measurement

[0072] A plate reader (TECAN, infinite F500) was used for measurement of E2 by the following method.

[0073] Anti-E2 rabbit monoclonal antibody was prepared as solid phase on a 96-well microplate (black, Greiner) in an amount of 0.25 μg/mL (carbonate buffer (0.05 M, pH 9.6)). Blocking treatment was then carried out with 1% skim milk/PBS. Using a separate plate, an E2 dilution series (2× dilution from 5,000 μg/mL) was prepared and dispensed into an anti-E2 rabbit monoclonal antibody-immobilized 96-well microplate at 100 μL/well. After incubating for 5 minutes, it was washed using a plate washer. Anti-immunocomplex mouse monoclonal antibody labeled with alkaline phosphatase was prepared to an absorbance of 1 mA at 280 nm, and then dispensed at 100 μL/well and incubated for 5 minutes. It was then washed using a plate washer, 4-MUP (4-methylumbelliferyl phosphate, Merck) was added as enzyme substrate and the fluorescence intensity was measured after 30 minutes.

[Comparative Example 1] Example Using Anti-Immunocomplex Antibody as Solid Phase Antibody

[0074] A Comparative Example of the invention will now be described in detail, for a method using rabbit monoclonal antibody for estradiol (E2) and anti-immunocomplex mouse monoclonal antibody for the immune complex, with the anti-immunocomplex mouse monoclonal antibody immobilized on a water-insoluble carrier (hereunder referred to as “solid phase antibody”), on a 96-well ELISA plate.

(1) Anti-E2 Rabbit Monoclonal Antibody

[0075] The anti-E2 rabbit monoclonal antibody used was the same antibody as in Example 1.

(2) Anti-Immunocomplex Mouse Monoclonal Antibody

[0076] The anti-immunocomplex mouse monoclonal antibody used was the same antibody as in Example 1.

(3) Measurement

[0077] A plate reader (TECAN, infinite F500) was used for measurement of E2 by the following method.

[0078] Anti-immunocomplex mouse monoclonal antibody was prepared as solid phase on a 96-well microplate (black, Greiner) in an amount of 0.25 μg/mL (carbonate buffer (0.05 M, pH 9.6)). Blocking treatment was then carried out with 1% skim milk/PBS. Using a separate plate, an E2 dilution series (2× dilution from 5,000 μg/mL final concentration) was prepared, and alkaline phosphatase-labeled anti-E2 rabbit monoclonal antibody was added to a final concentration of 1 mA (absorbance at 280 nm), before immediate dispensing into an anti-immunocomplex mouse monoclonal antibody-immobilized 96-well microplate at 100 μL/well. After incubation for 10 minutes, it was washed using a plate washer, 4-MUP (4-methylumbelliferyl phosphate, Merck) was added as enzyme substrate and the fluorescence intensity was measured after 30 minutes.

[0079] Table 3 below shows the measurement results for fluorescence intensity in Example 3 and Comparative Example 1. For easier comparison, Table 4 and FIG. 4 show the S/N (signal/noise) ratio calculation results. For all E2 concentrations, S/N was larger in Example 3 (anti-E2 rabbit monoclonal antibody as solid phase antibody), confirming that measurement sensitivity was higher when using anti-E2 rabbit monoclonal antibody as the solid phase antibody, even with the same antibody combination.

TABLE-US-00003 TABLE 3 E2 concentration (pg/mL) 0 10 20 39 78 156 313 625 1,250 2,500 5,000 Example 3 (anti-E2 2,026 4,461 6,419 9,493 14,019 19,296 23,181 23,613 24,534 24,459 25,828 antibody solid phase) Comp. Ex. 1 (anti- 2,044 2,577 3,250 3,766 9,560 12,350 15,434 14,531 13,968 14,008 13,419 immunocomplex antibody solid phase)

TABLE-US-00004 TABLE 4 E2 concentration (pg/mL) 10 20 39 78 156 313 625 1,250 2,500 5,000 Example 3 (anti-E2 2.2 3.2 4.7 6.9 9.5 11.4 11.7 12.1 12.1 12.7 antibody solid phase) Comp. Ex. 1 (anti- 1.3 1.6 1.8 4.7 6.0 7.6 7.1 6.8 6.9 6.6 immunocomplex antibody solid phase)

Example 4

[0080] An Example of the present invention will now be described in detail, for a method using rabbit monoclonal antibody for digoxin (Dig) and anti-immunocomplex mouse monoclonal antibody for the immune complex, with the understanding that the Example is not intended to be restrictive on the invention.

(1) Anti-Dig Rabbit Monoclonal Antibody

[0081] The anti-Dig rabbit monoclonal antibody was obtained by the method described in Japanese Unexamined Patent Publication No. 2009-240300.

(2) Anti-Immunocomplex Mouse Monoclonal Antibody

[0082] The antibody for Dig/anti-Dig rabbit monoclonal antibody immune complex (anti-immunocomplex mouse monoclonal antibody) was obtained by the method described in Japanese Patent Publication No. 6031944.

(3) Measurement

[0083] Automatic analysis was carried out by the following method using a fully automatic chemiluminescent enzyme immunoassay device (AIA-CL2400 by Tosoh Corp.).

(3-1) Preparation of Reagent Cup

[0084] A cup having two cells as shown in FIG. 1 was used to prepare a reagent cup for use in automatic analysis. A solution containing microparticles immobilizing anti-Dig rabbit monoclonal antibody was dispensed in an amount of 50 μL into the microparticle cell. A solution containing alkaline phosphatase-labeled anti-immunocomplex mouse monoclonal antibody was dispensed in an amount of 75 μL into the conjugate cell. This was sealed with aluminum to obtain a reagent cup for Dig assay.

(3-2) Measurement

[0085] The Dig-assay reagent cup prepared in (3-1) was set in the fully automatic chemiluminescent enzyme immunoassay device (AIA-CL2400 by Tosoh Corp.) and measurement was conducted for 6 different samples (cal1 to cal6) of known Dig concentration to construct a calibration curve. For the measurement, 10 μL of a sample of known concentration and 40 μL of a diluent were dispensed into the microparticle cell of the Dig-assay reagent cup and reacted for 5 minutes (first-order reaction), after which washing was performed with a cleaning fluid (B/F separation) and the reagent in the conjugate cell (50 μL) was transferred to the microparticle cell and reacted (secondary reaction, 3 minutes). After completion of the secondary reaction, a second washing was performed with cleaning fluid. Enzyme substrate was then added and the emission intensity was measured. The measurement results are shown in Table 5 and FIG. 5.

[0086] The emission intensity was confirmed to increase in proportion to Dig concentration, making it possible to construct a Dig assay system using anti-immunocomplex antibody.

TABLE-US-00005 TABLE 5 Dig concentration (ng/mL) 0.0 0.5 1.0 2.0 3.0 4.0 Emission 657 15,225 32,354 66,991 97,732 117,363 intensity (cps)

[0087] These results confirmed that Dig can be detected in an assay system using anti-hapten rabbit monoclonal antibody as a solid phase antibody and anti-immunocomplex antibody as a labeled antibody.

Example 5

[0088] An Example of the invention will now be described in detail, for rabbit monoclonal antibody for estradiol (E2) and anti-immunocomplex mouse monoclonal antibody for the immune complex, and an absorption antibody (EE2B-18) added to reduce cross-reactivity, with the understanding that this Example is not restrictive on the invention.

(1) Anti-E2 Rabbit Monoclonal Antibody

[0089] The anti-E2 rabbit monoclonal antibody used was the same antibody as in Example 1.

(2) Anti-Immunocomplex Mouse Monoclonal Antibody

[0090] The anti-immunocomplex mouse monoclonal antibody used was the same antibody as in Example 1.

(3) Absorption Antibody (Antibody for Ethynylestradiol (EE2))

[0091] The absorption antibody, i.e. antibody for the E2 analog EE2 (EE2B-18) was obtained by the following method.

(3-1) Immunization of Animals

[0092] The immunized animals used were six 5-week-old female mice. The antigen used was 1,3,5(10)-estratrien-17-α-ethynyl-3,17-beta-diol-6-one-6-carboxymethyloxime:BSA (Steraloids Co.), and an emulsion comprising the antigen solution and an adjuvant in equal amounts was prepared and used to immunize the mice 4 times at 1 week intervals. The immunization amount was 50 μg of antigen per mouse. The adjuvant used was Freund's complete adjuvant for the first inoculation, and Freund's incomplete adjuvant for the second and subsequent inoculations.

(3-2) Confirmation of Antibody Titer

[0093] The increase in antibody titer was confirmed by ELISA as described below.

(3-2-1)

[0094] EE2 chemically bound to KLH (KLH-EE2) was immobilized on an ELISA plate at 1 μg/mL and blocked with 1% skim milk solution.

(3-2-2)

[0095] The obtained mouse antiserum was used to prepare a dilution series (2× dilution) from 1000× dilution and reacted with the KLH-EE2 in solid-phase on the ELISA plate.

(3-2-3)

[0096] After B/F (Bound/Free) separation, α-Mouse IgG-ALP (Merck) was added to the plate as an alkaline phosphatase (ALP)-labeled antibody, and reacted with the mouse antibody on the plate.

(3-2-4)

[0097] After B/F separation of the unreacted ALP-labeled antibody, the ALP substrate 4-methylumbelliferylphosphoric acid (4-MUP) was dispensed into the plate, the fluorescence intensity was measured to detect it, and the mice with increased antibody titer were selected.

(3-3) Creation of Absorption Antibody-Producing Hybridomas

[0098] Antibody producing cells were prepared from the mice selected in (3-2) by the following method.

(3-3-1) Spleens were extracted from mice with increased antibody titer, and spleen cells were prepared by an established method. The prepared spleen cells were fused with mouse myeloma cells by electrical fusion to create hybridomas.
(3-3-2) The fused hybridoma suspensions were suspended in E-RDF medium (Kyokuto Pharmaceutical Industrial Co., Ltd.) containing 10% FCS (Fetal calf serum) and 1×HAT (Sigma), and then spread on a microtiter plate and cultured for 8 days, after which the culture supernatants were obtained.

(3-4) Screening of Absorption Antibody-Producing Hybridomas

[0099] The absorption antibody preferably has high reactivity with EE2 and low reactivity with E2. For screening of the absorption antibody of the invention, therefore, it is sufficient to select antibodies that selectively react with EE2 even in the presence of E2. Specifically, the screening was carried out by ELISA in the following manner.

(3-4-1)

[0100] KLH-EE2 was immobilized on an ELISA plate at 1 μg/mL and blocked with 1% skim milk solution.

(3-4-2)

[0101] The culture supernatant was reacted with KLH-EE2 in solid phase on ELISA plates in the presence or in the absence of E2.

(3-4-3)

[0102] After B/F separation, α-Mouse IgG-ALP (Merck) was added to the plate as ALP-labeled antibody, and reacted with the mouse antibody on the plate.

(3-4-4)

[0103] The unreacted ALP-labeled antibody was removed by B/F separation, after which the ALP substrate 4-MUP was dispensed into the plate and detected by fluorescence intensity measurement, and hybridomas producing antibodies that reacted with EE2 in the presence of E2 were selected out.

(3-5) Production and Purification of Absorption Antibodies

[0104] The absorption antibody-producing hybridomas selected in (3-4) were cultured in E-RDF medium (Kyokuto Pharmaceutical Industrial Co., Ltd.) containing 10% FCS, and the culture supernatants were obtained. The antibodies in the culture supernatants were concentrated by ammonium sulfate precipitation and a TSKgel Ether-5pw column was used for purification to obtain an absorption antibody (EE2B-18).

(4) Automatic Analysis

[0105] Automatic analysis was carried out by the following method using a fully automatic chemiluminescent enzyme immunoassay device (AIA-CL2400 by Tosoh Corp.).

(4-1) Preparation of Reagent Cup

[0106] A cup having two cells was used to prepare a reagent cup for use in automatic analysis. Absorption antibody (EE2B-18) was added at 10 μg/mL to a solution containing microparticles immobilizing anti-E2 rabbit monoclonal antibody, and the mixture was dispensed into the microparticle cell. A solution containing β-estradiol-6-one-6-(O-carboxymethyloxime) was dispensed into the conjugate cell together with alkaline phosphatase-labeled anti-immunocomplex antibody, with reference to PTL 1. The solution was lyophilized and sealed with aluminum to prepare an E2-assay reagent cup.

(4-2) Measurement

[0107] The E2-assay reagent cup prepared in (4-1) was set in the fully automatic chemiluminescent enzyme immunoassay device (AIA-CL2400 by Tosoh Corp.) and measurement was conducted for 6 different samples (cal1 to cal6) of known E2 concentration to construct a calibration curve. The measurement was carried out in the same manner as (3-2) of Example 1. The measurement results are shown in FIG. 6. The emission intensity was confirmed to increase in proportion to E2 concentration, making it possible to construct an automatic analysis system for E2 using anti-immunocomplex antibody when in the presence of absorption antibody.

(4-3) Evaluation of Cross-Reactivity

[0108] A solution prepared to 10 ng/mL EE2 was used for measurement of the emission intensity in the same manner as (4-2), using the E2-assay reagent cup prepared in (4-1) and AIA-CL2400. The cross-reaction rate was calculated from the calibration curve prepared in (4-2). The results are shown in Table 6.

Example 6

[0109] An example containing no absorption antibody (EE2B-18) in the E2-assay reagent cup prepared in Example 5 will now be described as Example 6.

(1) E2-Assay Reagent Cup Containing No Absorption Antibody

[0110] An E2-assay reagent cup was produced in the same manner as Example 5(4-1), but without addition of absorption antibody.

(2) Measurement

[0111] The E2-assay reagent cup prepared in (1) was set in an AIA-CL2400 and measurement was conducted for 6 different samples (cal1 to cal6) of known E2 concentration to construct a calibration curve. The measurement was carried out in the same manner as (3-2) of Example 1. The measurement results are shown in FIG. 7. It was confirmed that with or without absorption antibody, there was no significant change in the calibration curve with respect to FIG. 6 (Example 5).

(3) Evaluation of Cross-Reactivity

[0112] Measurement was carried out in the same manner as Example 5(4-2) with an AIA-CL2400, using the E2-assay reagent cup prepared in (1) and a solution prepared to 10 ng/mL EE2. The cross-reaction rate was calculated from the calibration curve prepared in (2). The cross-reaction rates measured in Example 5 and Example 6 are shown in Table 6.

TABLE-US-00006 TABLE 6 Example 5 Example 6 Cross-reaction rate (%) 9.7 15.4

[0113] The results for the cross-reaction rates in Table 6 indicate that the presence of absorption antibody (EE2B-18) can drastically improve cross-reaction. This confirmed that the presence of absorption antibody can inhibit cross-reaction in an assay system using anti-immunocomplex antibody.

Example 7

[0114] An Example of the invention will now be described in detail, for rabbit monoclonal antibody for estradiol (E2) and anti-immunocomplex mouse monoclonal antibody for the immune complex, and an absorption antibody added to reduce cross-reactivity, with the understanding that this Example is not restrictive on the invention.

(1) Anti-E2 Rabbit Monoclonal Antibody

[0115] The anti-E2 rabbit monoclonal antibody was obtained by the method described in Japanese Unexamined Patent Publication No. 2009-240300.

(2) Anti-Immunocomplex Mouse Monoclonal Antibody

[0116] The antibody for E2/anti-E2 rabbit monoclonal antibody immune complex (anti-immunocomplex mouse monoclonal antibody) was obtained by the method described in Japanese Patent Publication No. 6031944.

(3) Absorption Antibody (Antibody for Ethynylestradiol (EE2))

[0117] The antibody for the E2 analog EE2 was prepared by the same method as Example 5 as a different antibody from EE2B-18.

(4) Automatic Analysis

[0118] Automatic analysis was carried out by the following method using a fully automatic chemiluminescent enzyme immunoassay device (AIA-CL2400 by Tosoh Corp.).

(4-1) Preparation of Reagent Cup

[0119] A cup having two cells was used to prepare a reagent cup for use in automatic analysis.

[0120] Absorption antibody was added at 10 μg/mL to a solution containing microparticles immobilizing anti-E2 rabbit monoclonal antibody, and the mixture was dispensed into the microparticle cell. A solution containing β-estradiol-6-one-6-(O-carboxymethyloxime) was dispensed into the conjugate cell together with alkaline phosphatase-labeled anti-immunocomplex antibody, with reference to PTL 1. The solution was lyophilized and scaled with aluminum to prepare an E2-assay reagent cup.

(4-2) Measurement

[0121] The E2-assay reagent cup prepared in (4-1) was set in the fully automatic chemiluminescent enzyme immunoassay device (AIA-CL2400 by Tosoh Corp.) and measurement was conducted for 6 different samples (cal1 to cal6) of known E2 concentration to construct a calibration curve. The measurement results are shown in FIG. 8. The emission intensity was confirmed to increase in proportion to E2 concentration, making it possible to construct an automatic analysis system for E2 using anti-immunocomplex antibody when in the presence of absorption antibody.

(4-3) Evaluation of Cross-Reactivity

[0122] A solution prepared to 10 ng/mL EE2 was used for measurement of the emission intensity using the E2-assay reagent cup prepared in (4-1) and AIA-CL2400. The cross-reaction rate was calculated from the calibration curve prepared in (4-2). The results are shown in Table 7.

Example 8

[0123] An example containing no absorption antibody in the E2-assay reagent cup prepared in Example 7 will now be described as Example 8.

(1) E2-Assay Reagent Cup Containing No Absorption Antibody

[0124] An E2-assay reagent cup was produced in the same manner as Example 7(4-1), but without addition of absorption antibody.

(2) Measurement

[0125] The E2-assay reagent cup prepared in (1) was set in an AIA-CL2400 and measurement was conducted for 6 different samples (cal1 to cal6) of known E2 concentration to construct a calibration curve. The measurement results are shown in FIG. 9. It was confirmed that with or without absorption antibody, there was no significant change in the calibration curve with respect to FIG. 8 (Example 7).

(3) Evaluation of Cross-Reactivity

[0126] Measurement was carried out with an AIA-CL2400, using the E2-assay reagent cup prepared in (1) and a solution prepared to 10 ng/mL EE2. The cross-reaction rate was calculated from the calibration curve prepared in (2).

[0127] The cross-reaction rates measured in Example 7 and Example 8 are shown in Table 7.

TABLE-US-00007 TABLE 7 Example 7 Example 8 Cross-reaction rate (%) 6.0 14.9

[0128] The results for the cross-reaction rates indicate that the presence of absorption antibody can drastically improve cross-reaction. This confirmed that the method of the invention can inhibit cross-reaction in an assay system using anti-immunocomplex antibody.

[0129] The present invention has been described in detail using specific embodiments, but it will be apparent to a person skilled in the art that various modifications and alterations may be employed such as are within the spirit and scope of the invention.

[0130] The entire contents of the specifications, claims, drawings and abstracts of Japanese Patent Application No. 2019-209407 filed on Nov. 20, 2019, Japanese Patent Application No. 2019-216521 filed on Nov. 29, 2019, Japanese Patent Application No. 2020-042915 filed on Mar. 12, 2020, Japanese Patent Application No. 2020-100842 filed on Jun. 10, 2020 and Japanese Patent Application No. 2020-138101 filed on Aug. 18, 2020, are incorporated by reference in the present disclosure.