Measurement method using anti-immunocomplex antibody

Abstract

To provide a measurement method capable of increasing the reaction efficiency in a measurement system using an anti-immuno-complex antibody.SOLUTION: An immunoassay method using an anti-hapten rabbit monoclonal antibody immobilized on a water-insoluble carrier and a labeled anti-immunocomplex antibody comprises the following steps (i)-(iii): (i) reacting the anti-hapten rabbit monoclonal antibody immobilized on the water-insoluble carrier with a hapten in a solution to be measured, (ii) reacting the labeled anti-immunocomplex antibody with an immune complex of the hapten-anti-hapten rabbit monoclonal antibody, and (iii) detecting a signal derived from the labeling.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a measurement method using an anti-immunocomplex antibody. [Background technology]

[0002] Haptens, which are low molecular weight compounds, are usually measured by an immunoassay method called a competitive method. The competitive method is a method in which a hapten contained in a sample and a labeled hapten labeled with a radioisotope or an enzyme are competitively reacted with a certain amount of anti-hapten antibody. As the amount of hapten contained in a sample increases, the amount of the 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 the 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 a very small amount of hapten (Non-Patent Document 1).

[0003] 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) Immobilize an anti-immunocomplex antibody on a plate or the like. (2) A sample containing the target hapten is mixed with an enzyme-labeled anti-hapten antibody 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.

[0004] A specific example of a measurement system using an anti-immunocomplex antibody is an estradiol (E2) measurement system as 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 in advance, and then reacting the mixture with a carrier on which the anti-immunocomplex antibody is immobilized.

[0005] 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, that is, a solid-liquid interface, and therefore 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 considered that the overall reaction efficiency is reduced.

[0006] As described in Non-Patent Document 1, a measurement system in which a labeled anti-hapten antibody and a measurement target are mixed first, 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 described in Non-Patent Document 3, there are cases in which a false high value or a false low value is reported in a one-step measurement due to the presence of coexisting substances in the measurement target liquid. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Analysis, 551-552;2004 [Non-Patent Document 2] 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 method for measuring antigen-antibody reactions [P3093] [Non-Patent Document 3] Yasuteru Adachi, Hoki Ogawa, Nami Tosaoka, Kenichi Mukaiyama Abstract of the 67th Annual Meeting of the Japanese Society of Medical Technology (May 2018) [Non-Patent Document 4] Junichi Takagi, "Analysis of intermolecular interactions of proteins," Understanding the functions of proteins - molecular functions and biological effects, Kagaku Dojin, 2009, p.29-51 [Patent documents]

[0008] [Patent Document 1] Patent No. 6221466 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a measurement method capable of increasing the reaction efficiency in a measurement system using an anti-immunocomplex antibody. [Means for solving the problem]

[0010] 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), 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), 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 the step (i) is carried out in the presence of an absorbing antibody.

[0011] The present invention will be described in detail below.

[0012] (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 them, E2 and Dig are more preferred. Among thyroid hormones, thyroxine is preferred.

[0013] (2) Measurement target solution The solution to be measured is not particularly limited as long as it contains the hapten to be measured, and is preferably a body fluid such as human blood (whole blood, plasma, serum, etc.) or urine.

[0014] (3) Anti-immunocomplex antibodies The 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 the animal species include mouse, rat, goat, sheep, etc. In the present invention, it is particularly preferable to obtain the anti-immunocomplex monoclonal antibody from a mouse.

[0015] (4) Anti-hapten rabbit monoclonal antibody In the present invention, a rabbit monoclonal antibody is used as the anti-hapten antibody. The reason is that a rabbit monoclonal antibody has a higher affinity for a hapten than a mouse monoclonal antibody. The affinity is a dissociation constant (KD) of 10 -10 (M) or less, and more preferably 10 -11 It is preferable that the thickness is equal to or less than (M).

[0016] In particular, in the present invention, since an anti-hapten antibody is used as the solid-phase antibody, it is essential to use 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 difficult to rebind to the antibody on the solid-phase surface. Thus, in the present invention, in order to suppress dissociation of the hapten from the solid-phase antibody, it is essential to use a rabbit monoclonal antibody with high affinity as the solid-phase antibody.

[0017] (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 (e.g., via avidin-biotin binding, etc.).

[0018] (6)Signs 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 the labeling may be direct or indirect (for example, via avidin-biotin binding, etc.).

[0019] (7) Process (i)~(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.

[0020] (8) Cleaning process The washing step is a step of washing away components nonspecifically adsorbed to the water-insoluble carrier. When measuring a solution to be measured that contains 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 did not function properly due to the presence of coexisting substances. Even in such cases, the influence of the coexisting substances can be reduced by carrying out the 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).

[0021] (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 Figure 1. (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 test solution in the automated immunoassay device. (9-3) In an automated immunoassay device, a solution to be measured containing a hapten is dispensed into a cell on the water-insoluble carrier side of the 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) The enzyme substrate is added, and the luminescence intensity of the product resulting from the enzyme reaction is measured. (10) Cross-reaction Cross-reaction is a reaction between a substance similar to the substance to be measured (a cross-reactive substance) and 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. Another cause was considered to be 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.

[0022] (11) Absorption of antibodies The absorbing antibody is an antibody that hardly reacts with the hapten to be measured and specifically reacts with the cross-reactive substance. It is preferable that the absorbing antibody has no reactivity with the hapten to be measured, but there is no problem if the absorbing antibody has a weak reactivity within a range that does not affect the measurement sensitivity. Specifically, the reactivity of the absorbing antibody with the hapten to be measured is preferably about 1 / 100 or less of the reactivity with the cross-reactive substance. Effect of the Invention

[0023] 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, and it is possible to provide a measurement system in which the influence of coexisting substances is reduced. Furthermore, when an absorbing antibody is coexisted 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. [Brief description of the drawings]

[0024] [Figure 1] FIG. 2 is a diagram showing an example of a reagent cup that can be used in the present invention. [Diagram 2] FIG. 1 shows the results of the calibration curve in Example 1 (3-2). [Diagram 3] FIG. 13 is a diagram showing the results of the calibration curve in Example 2 (3-2). [Figure 4] FIG. 1 is a diagram showing the S / N ratios of Example 3 and Comparative Example 1. [Diagram 5] FIG. 13 is a diagram showing the results of the calibration curve in Example 4 (3-2). [Figure 6] FIG. 13 is a diagram showing the results of the calibration curve in Example 5 (4-2). [Figure 7] FIG. 13 is a diagram showing the results of the calibration curve in Example 6(2). EXAMPLES

[0025] [Example 1] Below, 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.

[0026] (1) Anti-E2 rabbit monoclonal antibody The anti-E2 rabbit monoclonal antibody was obtained by the method described in JP-A-2009-240300.

[0027] (2) Anti-immunocomplex mouse monoclonal antibody The 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.

[0028] (3) Measurement The automated analysis was carried out using a fully automated chemiluminescent enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation) according to the following method.

[0029] (3-1) Preparation of reagent cup A reagent cup for automatic analysis was prepared using a cup with two cells as shown in Figure 1. (Hereinafter, one cell of the cup will be referred to as the particle 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.

[0030] (3-2) Measurement The E2 measurement reagent cup prepared in (3-1) was set in a fully automated chemiluminescence enzyme immunoassay device (AIA-CL2400, Tosoh Corporation), and six samples with known E2 concentrations (cal1 to cal6) were measured to create a calibration curve. For the measurement, 50 μL of the sample with known concentration and 50 μL of diluent were dispensed into the cell on the particle 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 the reagent (50 μL) in the cell on the conjugate side dissolved in the diluent was transferred to the cell on the particle side and reacted (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 Figure 2. It was confirmed that the luminescence intensity increased depending on the E2 concentration, confirming that an E2 measurement system using anti-immunocomplex antibodies had been constructed.

[0031] [Table 1]

[0032] From the above results, it was confirmed that E2 can be detected with high sensitivity 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.

[0033] [Example 2] Below, 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 thyroxine (FT4) and an anti-immunocomplex mouse monoclonal antibody against the immune complex thereof, but the present invention is not limited thereto.

[0034] (1) Anti-FT4 rabbit monoclonal antibody The anti-FT4 rabbit monoclonal antibody was obtained by the method described in JP-A-2009-240300.

[0035] (2) Anti-immunocomplex mouse monoclonal antibody The 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.

[0036] (3) Measurement The automated analysis was carried out using a fully automated chemiluminescent enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation) according to the following method.

[0037] (3-1) Preparation of reagent cup A reagent cup for automatic analysis was prepared using a cup with 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.

[0038] (3-2) Measurement The FT4 measurement reagent cup prepared in (3-1) was set in a fully automated chemiluminescence enzyme immunoassay device (AIA-CL2400, Tosoh Corporation), and six samples with known FT4 concentrations (cal1 to cal6) were measured to create a calibration curve. For the measurement, 5 μL of the sample with known concentration and 5 μL of the dilution solution were dispensed into the cell on the particle side of the FT4 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) from the conjugate side cell to the particle 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 2 and Figure 3. It was confirmed that the luminescence intensity increased depending on the concentration of FT4, confirming that a measurement system for FT4 using anti-immunocomplex antibodies had been constructed.

[0039] [Table 2]

[0040] From the above results, it was confirmed that FT4 can be detected with high sensitivity 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.

[0041] [Example 3] Hereinafter, an example of the present invention will be described in detail with respect to a method 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.

[0042] (1) Anti-E2 rabbit monoclonal antibody The anti-E2 rabbit monoclonal antibody used was the same as that described in Example 1.

[0043] (2) Anti-immunocomplex mouse monoclonal antibody The anti-immunocomplex mouse monoclonal antibody used was the same as that described in Example 1.

[0044] (3) Measurement E2 was measured using a plate reader (TECAN, infinite F500) according to the following method.

[0045] Anti-E2 rabbit monoclonal antibody was immobilized on a 96-well microplate (black, Greiner) at 0.25 μg / mL (Carbonate Buffer (0.05M pH9.6)). Afterwards, 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 the anti-E2 rabbit monoclonal antibody was immobilized. After incubation for 5 minutes, washing was performed using a plate washer. Next, an 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. Thereafter, 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 30 minutes later.

[0046] [Comparative Example 1] Example in which anti-immunocomplex antibody was used as solid-phase antibody Below, as a comparative example of the present invention, a detailed description will be given of a case in which 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 a "solid-phase antibody") and carried out in a 96-well ELISA plate.

[0047] (1) Anti-E2 rabbit monoclonal antibody The anti-E2 rabbit monoclonal antibody used was the same as that described in Example 1.

[0048] (2) Anti-immunocomplex mouse monoclonal antibody The anti-immunocomplex mouse monoclonal antibody used was the same as that described in Example 1.

[0049] (3) Measurement E2 was measured using a plate reader (TECAN, infinite F500) according to the following method.

[0050] Anti-immunocomplex mouse monoclonal antibody was immobilized on a 96-well microplate (black, Greiner) at 0.25 μg / mL (Carbonate Buffer (0.05M pH9.6)). Afterwards, blocking treatment was performed with 1% skim milk / PBS solution. Using another plate, a dilution series of E2 (2-fold dilution from the final concentration of 5,000 pg / mL) was prepared, and anti-E2 rabbit monoclonal antibody labeled with alkaline phosphatase was added to the dilution series 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 the anti-immunocomplex mouse monoclonal antibody was immobilized. After incubation 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.

[0051] The fluorescence intensity measurement results of Example 3 and Comparative Example 1 are shown in Table 3. For ease of comparison, the S / N ratio (signal / noise) was calculated and the results are shown in Table 4 and Fig. 4. At all E2 concentrations, Example 3 (anti-E2 rabbit monoclonal antibody was 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.

[0052] [Table 3]

[0053] [Table 4]

[0054] [Example 4] The following provides a detailed description of an embodiment of the present invention 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.

[0055] (1) Anti-Dig rabbit monoclonal antibody Anti-Dig rabbit monoclonal antibody was obtained by the method described in JP 2009-240300 A.

[0056] (2) Anti-immunocomplex mouse monoclonal antibody The 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.

[0057] (3) Measurement The automated analysis was carried out using a fully automated chemiluminescent enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation) according to the following method.

[0058] (3-1) Preparation of reagent cup A reagent cup for automatic analysis was prepared using a cup with two cells as shown in Figure 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 placed to prepare the reagent cup for Dig measurement.

[0059] (3-2) Measurement The Dig measurement reagent cup prepared in (3-1) was set in a fully automated chemiluminescence enzyme immunoassay device (AIA-CL2400, Tosoh Corporation), and six samples with known Dig concentrations (cal1 to cal6) were measured to create a calibration curve. For the measurement, 10 μL of the sample with known concentration and 40 μL of the dilution solution were dispensed into the cell on the particle side of the Dig 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) from the conjugate side cell to the particle 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 5 and Figure 5. It was confirmed that the luminescence intensity increased depending on the Dig concentration, confirming that a Dig measurement system using anti-immunocomplex antibodies had been constructed.

[0060] [Table 5]

[0061] From the above results, it was confirmed that Dig can be detected in an assay system using anti-hapten rabbit monoclonal antibody as the solid-phase antibody and anti-immunocomplex antibody as the labeled antibody. [Example 5] Below, examples of the present invention will be described in detail 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 anti-E2 rabbit monoclonal antibody used was the antibody described in Example 1. (2) Anti-immunocomplex mouse monoclonal antibody The anti-immunocomplex mouse monoclonal antibody used was the antibody described in Example 1.

[0062] (3) Absorption antibodies (antibodies against ethinyl estradiol (EE2)) The absorbing antibody, that is, 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 (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. The immunization dose per mouse was 50μg of antigen. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second and subsequent immunizations.

[0063] (3-2) Confirmation of antibody titer The 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 mouse antisera obtained were diluted 1000-fold and then serially diluted (2-fold), 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 (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 antibodies, 4-methylumbelliferyl phosphate (4-MUP), an ALP substrate, was dispensed onto the plate and detected by measuring the fluorescence intensity, and mice with increased antibody titers were selected.

[0064] (3-3) Preparation of antibody-producing hybridomas Antibody-producing cells were produced 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) After fusion, the hybridoma suspension was suspended in E-RDF medium (Kyokuto Pharmaceutical) containing 10% FCS (fetal calf serum) and 1x HAT (Sigma), then plated on a microtiter plate and cultured for 8 days, and the culture supernatant was obtained.

[0065] (3-4) Screening of antibody-producing hybridomas It is preferable that the absorbing antibody reacts strongly with EE2 and has low reactivity with E2. Therefore, in screening for the absorbing antibody 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 antibodies, 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.

[0066] (3-5) Production and purification of absorbed antibodies The antibody-producing hybridomas selected in (3-4) were 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 antibody (EE2B-18).

[0067] (4) Automatic analysis The automated analysis was carried out using a fully automated chemiluminescent enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation) according to the following method.

[0068] (4-1) Preparation of reagent cup A reagent cup for automatic analysis was prepared using a cup with two cells. An absorbing antibody (EE2B-18) was added to a solution containing microparticles with immobilized anti-E2 rabbit monoclonal antibody at a concentration of 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.

[0069] (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.

[0070] (4-3) Evaluation of cross-reactivity Using a solution adjusted to 10 ng / mL of EE2, the luminescence intensity was measured in the same manner as in (4-2) using the E2 measurement reagent cup prepared in (4-1) and the AIA-CL2400. The cross-reaction rate was calculated from the calibration curve prepared in (4-2). The results are shown in Table 6.

[0071] [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.

[0072] (1) Reagent cup for E2 measurement that does not contain absorbed antibodies A reagent cup for E2 measurement was prepared in the same manner as in Example 5 (4-1), except that no absorbing antibody was added.

[0073] (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 measurements were performed in the same manner as 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.

[0074] (3) Evaluation of cross-reactivity Using the E2 measurement reagent cup prepared in (1) and a solution adjusted to 10 ng / mL of EE2, measurements were performed in the same manner as in Example 5 (4-2) using the AIA-CL2400. The cross-reaction 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.

[0075] [Table 6]

[0076] The cross-reaction rate results in Table 6 show that the presence of the absorbed antibody (EE2B-18) can significantly improve cross-reaction. These results confirm that the presence of the absorbed antibody can suppress cross-reaction in a measurement system using an anti-immunocomplex antibody.

Claims

1. - An anti-hapten rabbit monoclonal antibody immobilized on a water-insoluble carrier, and - A labeled anti-immunocomplex antibody, An immunoassay method using the same, comprising the following steps (i) to (iii): (i) A step of reacting the anti-hapten rabbit monoclonal antibody immobilized on the water-insoluble carrier with the hapten in the measurement target solution, (ii) A step of reacting the labeled anti-immunocomplex antibody with the immune complex of the hapten-anti-hapten rabbit monoclonal antibody, (iii) A step of detecting the signal derived from the label, And the anti-immunocomplex antibody is a mouse monoclonal antibody, the method.

2. The method according to claim 1, wherein steps (i), (ii) and (iii) are performed in this order, and washing steps are provided between steps (i) and (ii) and between steps (ii) and (iii), respectively.

3. The method according to claim 1 or 2, wherein the label is alkaline phosphatase.

4. The method according to any one of claims 1 to 3, wherein the hapten is estradiol, thyroxine or digoxin.

5. The method according to any one of claims 1 to 4, wherein step (i) is performed in the presence of an absorption antibody.

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