JP7683188B2 - Measurement method using anti-immunocomplex antibody - Google Patents

Description

The present invention relates to a measurement method using an anti-immunocomplex antibody.

Haptens, which are low molecular weight compounds, are usually measured by an immunoassay method known as the competitive method. In the competitive method, a hapten contained in a sample and a labeled hapten labeled with a radioisotope or enzyme are made to react competitively with a certain amount of anti-hapten antibody. As the amount of hapten contained in a sample increases, the amount of labeled hapten that binds to the anti-hapten antibody decreases. From this, the amount of hapten contained in a sample can be estimated based on the ratio of labeled hapten that binds to the anti-hapten antibody. The measurement sensitivity in the competitive method depends on the affinity constant of the anti-hapten antibody used. However, it is difficult to obtain an anti-hapten antibody with a high affinity constant, and therefore it has been extremely difficult to measure very small amounts of hapten (Non-Patent Document 1).

As a measurement method that can solve the problems of such competitive immunoassays, a non-competitive immunoassay using an anti-immunocomplex antibody has been proposed. A hapten measurement system using an anti-immunocomplex antibody is carried out, for example, as shown below, and can measure haptens contained in a sample without relying on a competitive method.
(1) An anti-immunocomplex antibody is immobilized on a plate or the like.
(2) A sample containing a target hapten is mixed with an anti-hapten antibody labeled with an enzyme, and then added to (1).
(3) The excess of the labeled anti-hapten antibody is washed away and separated.
(4) A substrate for the labeling enzyme is added, and a signal resulting from the reaction between the enzyme and the substrate is detected.
Since the anti-immunocomplex antibody selectively binds to an immune complex consisting of a hapten and an anti-hapten antibody, an increase in the signal is observed as the amount of hapten contained in the sample increases.

A specific example of a measurement system using an anti-immunocomplex antibody is the estradiol (E2) measurement system shown in Patent Document 1. The technology disclosed in Patent Document 1 involves manually mixing an enzyme-labeled anti-E2 antibody with a measurement target liquid containing E2, and then reacting the mixture with a carrier on which the anti-immunocomplex antibody is immobilized.

In the method described in Patent Document 1, an anti-immunocomplex mouse monoclonal antibody is immobilized on a solid phase carrier. The reaction between the antibody immobilized on the solid phase and the antigen proceeds in a heterogeneous system at the solid-liquid interface, and the reaction efficiency is low (Non-Patent Document 2). When a low-affinity mouse monoclonal antibody is used as the solid phase, the reaction at the solid-liquid interface becomes a bottleneck, and it is thought that the overall reaction efficiency will decrease.

As described in Non-Patent Document 1, a measurement system in which a labeled anti-hapten antibody is first mixed with the measurement target, and then the resulting immune complex is reacted with an anti-immunocomplex antibody immobilized on a solid phase carrier is a one-step measurement that includes a washing step as one step. As described in Non-Patent Document 3, there are cases in which false high or low values are reported in one-step measurements due to the presence of coexisting substances in the measurement target liquid.

Analysis, 551-552; 2004 Nobuaki Muneta, Yuya Ishida, Masashi Kume, Toshihiko Imanami, Abstracts of the 53rd Annual Meeting of the Japan Society for Analytical Chemistry (2004) Development of a rapid and simple antigen-antibody reaction measurement method [P3093] Yasuteru Adachi, Hoki Ogawa, Nami Tosaoka, Kenichi Mukaiyama Abstract of the 67th Annual Meeting of the Japanese Society of Medical Technology (May 2018) Junichi Takagi, "Analysis of intermolecular interactions of proteins," Understanding the functions of proteins - molecular functions and biological actions, Kagaku Dojin, 2009, pp. 29-51

Patent No. 6221466

The object of the present invention is to provide a measurement method that can increase the reaction efficiency in a measurement system that uses an anti-immunocomplex antibody.

The present invention, made in view of the above problems, includes the following aspects.
(1) an anti-hapten rabbit monoclonal antibody immobilized on a water-insoluble carrier, and a labeled anti-immunocomplex antibody;
The method comprises the following steps (i) to (iii):
(i) reacting an anti-hapten rabbit monoclonal antibody immobilized on a water-insoluble carrier with a hapten in a solution to be measured;
(ii) reacting a labeled anti-immunocomplex antibody with the hapten-anti-hapten rabbit monoclonal antibody immune complex;
(iii) detecting a signal derived from the label.
(2) The method according to (1), in which steps (i), (ii) and (iii) are carried out in this order, and a washing step is provided between steps (i) and (ii), and between steps (ii) and (iii), respectively.
(3) The method according to (1) or (2), wherein the anti-immunocomplex antibody is a mouse monoclonal antibody.
(4) The method according to any one of (1) to (3), wherein the label is alkaline phosphatase.
(5) The method according to any one of (1) to (4), wherein the hapten is estradiol, thyroxine or digoxin.
(6) The method according to any one of (1) to (5), wherein step (i) is carried out in the presence of an absorbing antibody.

The present invention is described in detail below.

(1) Hapten The hapten in the present invention is not particularly limited as long as it is a substance with a small molecular weight that is usually measured by a competitive method, and examples thereof include thyroid hormones such as triiodothyronine, thyroxine, and 3,5-diiodo-L-thyronine, steroid hormones such as estrone, estradiol (E2), estriol, progesterone, and cortisol, and steroid glycosides such as digoxin (Dig). In particular, steroid hormones and steroid glycosides are preferred in the present invention, and among these, E2 and Dig are more preferred. Among thyroid hormones, thyroxine is preferred.

(2) Measurement target solution The measurement target solution is not particularly limited as long as it contains the measurement target hapten. For example, a body fluid such as human blood (whole blood, plasma, serum, etc.) or urine is preferable.

(3) Anti-immunocomplex antibody An anti-immunocomplex antibody is neither an antibody against a hapten nor an antibody against an antibody against a hapten, but an antibody against a complex of a hapten and an antibody against the hapten. In the present invention, the anti-immunocomplex antibody is preferably obtained from an animal species other than rabbit, from the viewpoint of obtaining an antibody specific to a complex of an anti-hapten rabbit monoclonal antibody and a hapten. Examples of animal species include mouse, rat, goat, sheep, etc. In the present invention, it is particularly preferable to obtain an anti-immunocomplex monoclonal antibody from a mouse.

(4) Anti-hapten rabbit monoclonal antibody In the present invention, a rabbit monoclonal antibody is used as the anti-hapten antibody. This is because rabbit monoclonal antibodies have a higher affinity for haptens than mouse monoclonal antibodies. The affinity is preferably such that the dissociation constant (KD) is 10 ^-10 (M) or less, more preferably 10 ^-11 (M) or less.

In particular, in the present invention, since an anti-hapten antibody is used as the solid-phase antibody, it is essential that the antibody is a rabbit monoclonal antibody with high affinity. As described in Non-Patent Document 4, an antigen once bound to an antibody on a solid-phase surface can rebind to a nearby solid-phase antibody even if it temporarily dissociates due to a washing operation or the like, if it is a protein (polymer) with a slow diffusion rate. However, since the antigen in the present invention is a low-molecular-weight hapten, once it dissociates from the antibody due to a washing operation or the like, it has a fast diffusion rate and is unlikely to rebind to the antibody on the solid-phase surface. Thus, in the present invention, in order to prevent the hapten from dissociating from the solid-phase antibody, it is essential that a rabbit monoclonal antibody with high affinity is used as the solid-phase antibody.

(5) Water-insoluble carrier In the present invention, the water-insoluble carrier is not particularly limited, and examples thereof include fine particles, particles, beads, plates, etc. of resin, glass, polystyrene, etc. Anti-hapten rabbit monoclonal antibodies are immobilized on the water-insoluble carrier. There is no particular limit to the immobilization method, and the antibody may be immobilized directly or indirectly (for example, via avidin-biotin bond, etc.).

(6) Labeling In the present invention, the label is not particularly limited, but for example, an enzyme, a radioisotope, a dye, etc. can be used. As the enzyme, alkaline phosphatase, etc. can be preferably used. The label is bound to an anti-immunocomplex antibody. There is no particular limit to the labeling method, and labeling may be performed directly or indirectly (for example, via avidin-biotin binding, etc.).

(7) Steps (i) to (iii)
The above steps (i) to (iii) are preferably carried out in this order, but steps (i) and (ii) may be carried out simultaneously. In addition, other steps may be present between steps (i) and (ii) or between steps (ii) and (iii). For example, by including a washing step described below between steps, the measurement sensitivity can be increased.

(8) Washing step The washing step is a step of washing away components nonspecifically adsorbed to the water-insoluble carrier. When measuring a solution containing a large amount of impurities such as blood, the antigen-antibody reaction may be inhibited by coexisting substances. There have also been reported cases where the measurement system does not function properly due to the presence of coexisting substances. Even in such cases, the influence of the coexisting substances can be reduced by performing a washing step. In order to reduce the influence of the coexisting substances, a 2-STEP method is particularly effective, which has a washing step between steps (i) and (ii) and between steps (ii) and (iii), i.e., a total of two washing steps (Non-Patent Document 3).

(9) Measurement method In the method of the present invention, it is preferable to use a fully automated immunoassay device such as that shown in Biological Sample Analysis, Vol. 39, No. 4 (2016). For example, it is preferable to perform the following automatic measurement using a reagent cup having two cells as shown in FIG.
(9-1) A water-insoluble carrier having an anti-hapten antibody immobilized thereon is dispensed into one cell of a two-cell cup, and an enzyme-labeled anti-immunocomplex antibody is dispensed into the other cell to form a reagent cup.
(9-2) Place the reagent cup and the solution to be measured in the automated immunoassay device.
(9-3) In an automatic immunoassay device, a solution to be measured containing a hapten is dispensed into a cell on the water-insoluble carrier side of a reagent cup and allowed to react.
(9-4) After the reaction is completed, the components not adsorbed to the water-insoluble carrier on which the anti-hapten antibody is immobilized are washed away.
(9-5) The enzyme-labeled anti-immunocomplex antibody in the reagent cup is transferred to the cell on the water-insoluble carrier side.
(9-6) After the reaction is completed, the components not adsorbed to the water-insoluble carrier on which the anti-hapten antibody is immobilized are washed away.
(9-7) A substrate for the enzyme is added, and the luminescence intensity of the product resulting from the enzyme reaction is measured.
(10) Cross reaction Cross reaction is a reaction of a substance similar to the substance to be measured (cross-reactive substance) with an antibody against the substance to be measured. When a measurement system with high cross-reactivity is used in an immunoassay reagent, the measurement result will be falsely high due to cross reaction. The present inventors have found that when a hapten that is usually measured using a competitive method is subjected to a sandwich-type assay using an anti-immunocomplex antibody, the sandwich-type assay may have a higher cross-reactivity. As a result of the inventors' investigation into this point, it was considered that one of the causes is that a larger number of anti-hapten antibodies are used in the sandwich-type assay than in the competitive method. In addition, it was also considered that another cause is that the surface structures of the immune complex between the anti-hapten antibody and the hapten and the complex between the anti-hapten antibody and the cross-reactive substance are the same, and the anti-immunocomplex antibody recognizes both.

(11) Absorbing antibody Absorbing antibodies are antibodies that hardly react with the hapten to be measured and specifically react with cross-reactive substances. It is preferable that they have no reactivity with the hapten to be measured, but there is no problem if they have weak reactivity within a range that does not affect the measurement sensitivity. Specifically, the reactivity of the absorbing antibody to the hapten to be measured is preferably about 1/100 or less of the reactivity to the cross-reactive substance.

When measuring a target hapten using an anti-immunocomplex antibody, a highly sensitive measurement system is desired. In the measurement method of the present invention, a water-insoluble carrier is used in which a high-affinity anti-hapten rabbit monoclonal antibody is immobilized on the solid-phase carrier. This makes it possible to reduce the decrease in reaction efficiency at the solid-liquid interface, which is considered to be inefficient. In addition, by using an anti-hapten antibody as the solid-phase antibody, it is possible to perform two or more washing steps as necessary after the reaction with the hapten contained in the solution to be measured, making it possible to provide a measurement system in which the influence of coexisting substances is reduced. Furthermore, if an absorbing antibody is coexistent during the reaction between the anti-hapten antibody and the hapten to be measured, it is possible to provide a measurement system in which cross-reaction is suppressed.

FIG. 2 is a diagram showing an example of a reagent cup that can be used in the present invention. FIG. 1 is a diagram showing the results of the calibration curve in Example 1 (3-2). FIG. 13 is a diagram showing the results of the calibration curve in Example 2 (3-2). FIG. 13 is a diagram showing the S/N ratios of Example 3 and Comparative Example 1. FIG. 13 is a diagram showing the results of the calibration curve in Example 4 (3-2). FIG. 13 is a diagram showing the results of the calibration curve in Example 5 (4-2). FIG. 13 is a diagram showing the results of the calibration curve in Example 6(2).

[Example 1]
Hereinafter, the examples of the present invention will be described in detail with respect to a method of carrying out the invention using a rabbit monoclonal antibody against estradiol (E2) and an anti-immunocomplex mouse monoclonal antibody against the immune complex thereof, but the present invention is not limited thereto.

(1) Anti-E2 Rabbit Monoclonal Antibody Anti-E2 rabbit monoclonal antibody was obtained by the method described in JP 2009-240300 A.

(2) Anti-immunocomplex mouse monoclonal antibody An antibody against the immune complex of E2 and anti-E2 rabbit monoclonal antibody (anti-immunocomplex mouse monoclonal antibody) was obtained by the method described in Japanese Patent No. 6,031,944.

(3) Measurement Automated analysis was performed using a fully automated chemiluminescent enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation) according to the following method.

(3-1) Preparation of Reagent Cup A reagent cup for automatic analysis was prepared using a cup having two cells as shown in Figure 1. (Hereinafter, one cell of the cup will be referred to as the microparticle side cell and the other cell as the conjugate side cell, according to the contents.)
A solution containing microparticles immobilized with anti-E2 rabbit monoclonal antibody was dispensed into the cell on the microparticle side. A solution containing an alkaline phosphatase-labeled anti-immunocomplex mouse monoclonal antibody and β-estradiol-6-one-6-(O-carboxymethyloxime) was dispensed into the cell on the conjugate side with reference to Patent Document 1. The solution was freeze-dried and sealed with aluminum to prepare a reagent cup for E2 measurement.

(3-2) Measurement The E2 measurement reagent cup prepared in (3-1) was set in a fully automated chemiluminescence enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation), and 6 types of samples with known E2 concentrations (cal1 to cal6) were measured to create a calibration curve. For the measurement, 50 μL of a sample with known concentration and 50 μL of dilution solution were dispensed into the cell on the microparticle side of the E2 measurement reagent cup, and reacted for 5 minutes (primary reaction), followed by washing with a washing solution (B/F separation), and then transferring the reagent (50 μL) of the conjugate side cell dissolved in the dilution solution to the microparticle side cell and reacting (secondary reaction, 3 minutes). After the secondary reaction was completed, a second wash with a washing solution was performed. Then, the enzyme substrate was added, and the luminescence intensity was measured. The measurement results are shown in Table 1 and FIG. 2.
It was confirmed that the luminescence intensity increased depending on the E2 concentration, and it was confirmed that an E2 measurement system using an anti-immunocomplex antibody had been established.

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

[Example 2]
The following provides a detailed explanation of the present invention with respect to an embodiment using a rabbit monoclonal antibody against thyroxine (FT4) and an anti-immunocomplex mouse monoclonal antibody against the immune complex thereof, but the present invention is not limited thereto.

(1) Anti-FT4 Rabbit Monoclonal Antibody Anti-FT4 rabbit monoclonal antibody was obtained by the method described in JP 2009-240300 A.

(2) Anti-immunocomplex mouse monoclonal antibody An antibody against the immune complex of FT4 and anti-FT4 rabbit monoclonal antibody (anti-immunocomplex mouse monoclonal antibody) was obtained by the method described in Japanese Patent No. 6,031,944.

(3) Measurement Automated analysis was performed using a fully automated chemiluminescent enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation) according to the following method.

(3-1) Preparation of Reagent Cup A reagent cup for automatic analysis was prepared using a cup having two cells as shown in Figure 1. 50 μL of a solution containing microparticles with immobilized anti-FT4 rabbit monoclonal antibody was dispensed into the cell on the microparticle side. 75 μL of a solution containing alkaline phosphatase-labeled anti-immunocomplex mouse monoclonal antibody was dispensed into the cell on the conjugate side. An aluminum seal was placed to prepare a reagent cup for FT4 measurement.

(3-2) Measurement The FT4 measurement reagent cup prepared in (3-1) was set in a fully automated chemiluminescence enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation), and six samples with known FT4 concentrations (cal1 to cal6) were measured to create a calibration curve. The measurement was carried out by dispensing 5 μL of the sample with known concentration and 5 μL of the dilution solution into the cell on the microparticle side of the FT4 measurement reagent cup, reacting for 5 minutes (primary reaction), washing with a washing solution (B/F separation), and then transferring the reagent (50 μL) from the conjugate side cell to the microparticle side cell and reacting (secondary reaction, 3 minutes). After the secondary reaction was completed, washing with a second washing solution was carried out. Then, the enzyme substrate was added and the luminescence intensity was measured. The measurement results are shown in Table 2 and FIG. 3.
It was confirmed that the luminescence intensity increased depending on the concentration of FT4, and it was confirmed that a measurement system for FT4 using an anti-immunocomplex antibody had been established.

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

[Example 3]
Hereinafter, the present invention will be described in detail with respect to an example thereof, which is carried out in a 96-well ELISA plate using a rabbit monoclonal antibody against estradiol (E2) and an anti-immunocomplex mouse monoclonal antibody against the immune complex thereof, but the present invention is not limited thereto.

(1) Anti-E2 Rabbit Monoclonal Antibody The same anti-E2 rabbit monoclonal antibody as that described in Example 1 was used.

(2) Anti-Immunocomplex Mouse Monoclonal Antibody The same antibody as that described in Example 1 was used as the anti-immunocomplex mouse monoclonal antibody.

(3) Measurement E2 was measured using a plate reader (TECAN, infinite F500) according to the following method.

Anti-E2 rabbit monoclonal antibody was immobilized on a 96-well microplate (black, Greiner) at 0.25 μg/mL (Carbonate Buffer (0.05 M pH 9.6)). After that, blocking treatment was performed with 1% skim milk/PBS solution. Using another plate, a dilution series of E2 (2-fold dilution from 5,000 pg/mL) was prepared and dispensed at 100 μL/well into the 96-well microplate on which anti-E2 rabbit monoclonal antibody was immobilized. After incubation for 5 minutes, washing was performed using a plate washer. Next, anti-immunocomplex mouse monoclonal antibody labeled with alkaline phosphatase was adjusted so that the absorbance at 280 nm was 1 mA, dispensed at 100 μL/well, and incubated for 5 minutes. After that, the plate was washed using a plate washer, and the enzyme substrate 4-MUP (4-methylumbelliferyl phosphate, MERCK) was added, and the fluorescence intensity was measured after 30 minutes.

Comparative Example 1 Example using anti-immunocomplex antibody as solid-phase antibody Hereinafter, as a comparative example of the present invention, a rabbit monoclonal antibody against estradiol (E2) and an anti-immunocomplex mouse monoclonal antibody against their immune complex are used, and the anti-immunocomplex mouse monoclonal antibody is used as an antibody immobilized on a water-insoluble carrier (hereinafter referred to as "solid-phase antibody"), and a case is carried out in a 96-well ELISA plate will be described in detail.

(1) Anti-E2 Rabbit Monoclonal Antibody The same anti-E2 rabbit monoclonal antibody as that described in Example 1 was used.

(2) Anti-Immunocomplex Mouse Monoclonal Antibody The same antibody as that described in Example 1 was used as the anti-immunocomplex mouse monoclonal antibody.

(3) Measurement E2 was measured using a plate reader (TECAN, infinite F500) according to the following method.

Anti-immunocomplex mouse monoclonal antibody was immobilized on a 96-well microplate (black, Greiner) at 0.25 μg/mL (Carbonate Buffer (0.05 M pH 9.6)). After that, blocking treatment was performed with 1% skim milk/PBS solution. Using another plate, a dilution series of E2 (2-fold dilution from a final concentration of 5,000 pg/mL) was prepared, to which alkaline phosphatase-labeled anti-E2 rabbit monoclonal antibody was added to a final concentration of 1 mA (absorbance at 280 nm), and immediately dispensed at 100 μL/well into the 96-well microplate on which anti-immunocomplex mouse monoclonal antibody was immobilized. After incubating for 10 minutes, the plate was washed using a plate washer, and the enzyme substrate 4-MUP (4-methylumbelliferyl phosphate, MERCK) was added, and the fluorescence intensity was measured after 30 minutes.

The results of measuring the fluorescence intensity in Example 3 and Comparative Example 1 are shown in Table 3. To facilitate comparison, the results of calculating the S/N ratio (signal/noise) are shown in Table 4 and Figure 4. At all E2 concentrations, Example 3 (anti-E2 rabbit monoclonal antibody is the solid-phase antibody) had a higher S/N ratio, and it was confirmed that even when the same antibody combination was used, the measurement sensitivity was higher when anti-E2 rabbit monoclonal antibody was used as the solid-phase antibody.

[Example 4]
Hereinafter, the present invention will be described in detail with respect to an embodiment using a rabbit monoclonal antibody against digoxin (Dig) and an anti-immunocomplex mouse monoclonal antibody against the immune complex thereof, but the present invention is not limited thereto.

(1) Anti-Dig Rabbit Monoclonal Antibody Anti-Dig rabbit monoclonal antibody was obtained by the method described in JP 2009-240300 A.

(2) Anti-Immunocomplex Mouse Monoclonal Antibody An antibody against the immune complex of Dig and anti-Dig rabbit monoclonal antibody (anti-immunocomplex mouse monoclonal antibody) was obtained by the method described in Japanese Patent No. 6,031,944.

(3) Measurement Automated analysis was performed using a fully automated chemiluminescent enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation) according to the following method.

(3-1) Preparation of Reagent Cup A reagent cup for automatic analysis was prepared using a cup having two cells as shown in FIG. 1. 50 μL of a solution containing microparticles with immobilized anti-Dig rabbit monoclonal antibody was dispensed into the cell on the microparticle side. 75 μL of a solution containing alkaline phosphatase-labeled anti-immunocomplex mouse monoclonal antibody was dispensed into the cell on the conjugate side. An aluminum seal was applied to prepare a reagent cup for Dig measurement.

(3-2) Measurement The Dig measurement reagent cup prepared in (3-1) was set in a fully automated chemiluminescence enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation), and measurements were performed on six samples with known Dig concentrations (cal1 to cal6), and a calibration curve was created. The measurement was performed by dispensing 10 μL of a sample with a known concentration and 40 μL of a dilution solution into the cell on the microparticle side of the Dig measurement reagent cup, reacting for 5 minutes (primary reaction), washing with a washing solution (B/F separation), and then transferring the reagent (50 μL) from the conjugate side cell to the microparticle side cell and reacting (secondary reaction, 3 minutes). After the secondary reaction was completed, washing with a second washing solution was performed. Then, the enzyme substrate was added, and the luminescence intensity was measured. The measurement results are shown in Table 5 and FIG. 5.
It was confirmed that the luminescence intensity increased depending on the Dig concentration, and it was confirmed that a Dig measurement system using an anti-immunocomplex antibody had been constructed.

From the above results, it was confirmed that Dig can be detected in an assay system using an anti-hapten rabbit monoclonal antibody as a solid-phase antibody and an anti-immunocomplex antibody as a labeled antibody.
[Example 5]
Examples of the present invention will be described in detail below with respect to a rabbit monoclonal antibody against estradiol (E2), an anti-immunocomplex mouse monoclonal antibody against their immune complex, and an absorbing antibody (EE2B-18) added for the purpose of reducing cross-reactivity, but the present invention is not limited to these.
(1) Anti-E2 Rabbit Monoclonal Antibody The antibody described in Example 1 was used as the anti-E2 rabbit monoclonal antibody.
(2) Anti-immunocomplex Mouse Monoclonal Antibody The antibody described in Example 1 was used as the anti-immunocomplex mouse monoclonal antibody.

(3) Absorbing antibody (antibody against ethinyl estradiol (EE2))
The absorbing antibody, ie, an antibody against EE2, an analogue of E2 (EE2B-18), was obtained by the method described below.
(3-1) Immunization of animals Six 5-week-old female mice were used as immunized animals. 1,3,5(10)-ESTRATRIEN-17α-ETHYNYL-3,17-beta-DIOL-6-ONE-6-CARBOXYMETHYLOXIME:BSA (manufactured by STERALOIDS) was used as the antigen, and an emulsion was prepared by mixing equal amounts of the antigen solution and the adjuvant, which was then immunized four times at weekly intervals against mice. The immunization dose per mouse was 50 μg as the antigen amount. As the adjuvant, Freund's complete adjuvant was used in the first immunization, and Freund's incomplete adjuvant was used in the second and subsequent immunizations.

(3-2) Confirmation of Antibody Titer An increase in antibody titer was confirmed by the ELISA shown below.
(3-2-1)
EE2 chemically bound to KLH (KLH-EE2) was immobilized on an ELISA plate at 1 μg/mL, and then blocked with a 1% skim milk solution.
(3-2-2)
The obtained mouse antisera was diluted 1000-fold to prepare a dilution series (2-fold dilution), and reacted with KLH-EE2 immobilized on an ELISA plate.
(3-2-3)
After B/F (Bound/Free) separation, αMouse IgG-ALP (Merck), an alkaline phosphatase (hereinafter referred to as ALP)-labeled antibody, was added to the plate and reacted with the mouse antibody on the plate.
(3-2-4)
After B/F separation of unreacted ALP-labeled antibody, 4-methylumbelliferyl phosphate (4-MUP), an ALP substrate, was dispensed onto the plate and detected by measuring the fluorescence intensity to select mice with elevated antibody titers.

(3-3) Preparation of Absorbed Antibody-Producing Hybridomas Antibody-producing cells were prepared from the mice selected in (3-2) by the method described below.
(3-3-1) The spleens of mice with elevated antibody titers were removed, and spleen cells were prepared according to a standard method. The prepared spleen cells were fused with mouse myeloma cells by electrofusion to produce hybridomas.
(3-3-2) The hybridoma suspension after fusion was suspended in E-RDF medium (Kyokuto Pharmaceutical Co., Ltd.) containing 10% FCS (fetal calf serum) and 1×HAT (Sigma), and then plated on a microtiter plate and cultured for 8 days, and the culture supernatant was obtained.

(3-4) Screening of hybridomas producing absorbing antibodies It is preferable that the absorbing antibodies react strongly with EE2 and have low reactivity with E2. Therefore, in screening of absorbing antibodies in the present invention, an antibody that selectively reacts with EE2 even in the presence of E2 may be selected. Specifically, screening was performed by the ELISA shown below.
(3-4-1)
KLH-EE2 was immobilized on an ELISA plate at 1 μg/mL, and then blocked with a 1% skim milk solution.
(3-4-2)
The culture supernatant was reacted with KLH-EE2 immobilized on an ELISA plate in the presence or absence of E2.
(3-4-3)
After B/F separation, αMouse IgG-ALP (Merck), an ALP-labeled antibody, was added to the plate and reacted with the mouse antibody on the plate.
(3-4-4)
After B/F separation of unreacted ALP-labeled antibody, 4-MUP was dispensed onto a plate and detected by measuring the fluorescence intensity to select hybridomas producing antibodies that react with EE2 in the presence of E2.

(3-5) Production and purification of absorbing antibody The absorbing antibody-producing hybridoma selected in (3-4) was cultured in E-RDF medium (Kyokuto Pharmaceutical Co., Ltd.) containing 10% FCS to obtain a culture supernatant. The antibody present in the culture supernatant was concentrated by ammonium sulfate precipitation and purified using a Tskgel Ether-5pw column to obtain an absorbing antibody (EE2B-18).

(4) Automated Analysis Automated analysis was carried out using a fully automated chemiluminescent enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation) according to the following method.

(4-1) Preparation of Reagent Cup A reagent cup for automatic analysis was prepared using a cup having two cells. An absorbing antibody (EE2B-18) was added to a solution containing microparticles on which anti-E2 rabbit monoclonal antibody was immobilized so as to be 10 μg/mL, and dispensed into the cell on the microparticle side. A solution containing an alkaline phosphatase-labeled anti-immunocomplex antibody and β-estradiol-6-one 6-(O-carboxymethyloxime) was dispensed into the cell on the conjugate side, with reference to Patent Document 1. The solution was freeze-dried and sealed with aluminum to prepare a reagent cup for E2 measurement.

(4-2) Measurement The E2 measurement reagent cup prepared in (4-1) was set in a fully automated chemiluminescent enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation), and six samples with known E2 concentrations (cal1 to cal6) were measured to create a calibration curve. The measurements were performed in the same manner as in the measurement in (3-2) of Example 1. The measurement results are shown in FIG. 6. It was confirmed that the luminescence intensity increased depending on the E2 concentration, and it was confirmed that an automatic analysis system for E2 using anti-immunocomplex antibodies could be constructed in the presence of absorbing antibodies.

(4-3) Evaluation of cross-reactivity Using a solution in which EE2 was adjusted to 10 ng/mL, the luminescence intensity was measured in the same manner as in (4-2) using the E2 measurement reagent cup prepared in (4-1) and AIA-CL2400. In addition, the cross-reactivity rate was calculated from the calibration curve prepared in (4-2). The results are shown in Table 6.

[Example 6]
Hereinafter, a case where the E2 measurement reagent cup prepared in Example 5 does not contain an absorbing antibody (EE2B-18) will be described as Example 6.

(1) Reagent cup for E2 measurement not containing absorbing antibody A reagent cup for E2 measurement was produced in the same manner as in Example 5 (4-1), except that no absorbing antibody was added.

(2) Measurement The E2 measurement reagent cup prepared in (1) was set in the AIA-CL2400, and six samples with known E2 concentrations (cal1 to cal6) were measured to create a calibration curve. The measurement was performed in the same manner as in the measurement in (3-2) of Example 1. The measurement results are shown in Figure 7. By comparing with Figure 6 (Example 5), it was confirmed that there was no significant change in the shape of the calibration curve with or without the absorption antibody.

(3) Evaluation of cross-reactivity Using the E2 measurement reagent cup prepared in (1) and a solution in which EE2 was adjusted to 10 ng/mL, measurements were performed in the same manner as in Example 5 (4-2) with an AIA-CL2400. The cross-reactivity rate was calculated from the calibration curve prepared in (2).
The cross-reaction rates measured in Examples 5 and 6 are shown in Table 6.

The cross-reaction rate results in Table 6 show that the presence of the absorbing antibody (EE2B-18) can significantly improve cross-reactions. These results confirm that the presence of the absorbing antibody can suppress cross-reactions in a measurement system using an anti-immunocomplex antibody.

Claims (5)

- an anti-hapten rabbit monoclonal antibody immobilized on a water-insoluble carrier, and - a labeled anti-immunocomplex antibody,
The immunoassay method includes the following steps (i) to (iii):
(i) reacting an anti-hapten rabbit monoclonal antibody immobilized on a water-insoluble carrier with a hapten in a solution to be measured;
(ii) reacting a labeled anti-immunocomplex antibody with the hapten-anti-hapten rabbit monoclonal antibody immune complex;
(iii) detecting a signal derived from the label ;
and wherein the anti-immunocomplex antibody is a mouse monoclonal antibody. The method according to claim 1, wherein steps (i), (ii) and (iii) are carried out in this order, and a washing step is provided between steps (i) and (ii) and between steps (ii) and (iii). The method of claim 1 or 2 , wherein the label is alkaline phosphatase. 4. The method according to claim 1, wherein the hapten is estradiol, thyroxine or digoxin. The method according to any one of claims 1 to 4 , wherein step (i) is carried out in the presence of an absorbing antibody.