JP3482853B2 - Hapten antigen-binding solid phase and immunoassay method using the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hapten antigen-binding solid phase and an immunoassay method using the same.

[0002]

2. Description of the Related Art As a method for measuring a hapten antigen,
The measurement by the competitive reaction method is widely used, and there are two methods of the competitive reaction method, a method using a solid-phase bound antigen and a method using a solid-phase bound antibody. In the former, in addition to directly binding the hapten antigen to the solid phase, the hapten antigen is bound to the antigen supporting substance such as a high molecular weight protein in advance, and then the hapten antigen-binding solid phase is bound to the solid phase by binding the antigen supporting substance to the solid phase. Is also used (Japanese Patent Laid-Open No. 64-686).
No. 61).

The competitive reaction method using the hapten antigen-bound solid phase is an antigen-antibody reaction in which an unknown amount of the antigen to be measured in the sample and a known amount of the immobilized antigen compete with the labeled antibody in the liquid phase. In this measuring method, the reaction concentration of each reactant is adjusted in order to obtain a good inhibition curve within the target measuring range. Generally, if the molar ratio of the immobilized hapten antigen to the hapten antigen to be measured is lowered, the sensitivity of the measurement system becomes higher. Therefore, by decreasing the amount of the hapten antigen bound to the solid phase, the relative molar ratio of the unknown amount of the antigen to the solid phase hapten antigen can be increased, and the measurement sensitivity can be increased.

However, reducing the amount of hapten antigen bound to the solid phase means reducing the amount of hapten antigen to the binding surface of the solid phase, and as a result, the hapten antigen non-binding surface on the solid phase increases. To do. In this case, if the masking of the hapten antigen non-binding surface is insufficient, serum-derived components such as lipids and various steroid-binding proteins,
In addition, components having an affinity for the hydrophobic surface of the solid phase are likely to be adsorbed. Such adsorption of the serum-derived component to the solid phase causes the antigen itself bound to the solid phase to be specifically or non-specifically coated with the serum-derived component, or the reaction between the labeled antibody and the immobilized antigen is steric. In some cases, the measured values were abnormally high, resulting in frequent results.

[0005]

Therefore, an object of the present invention is to provide a hapten antigen-bound solid phase capable of suppressing the influence of serum-derived components more than ever before in an immunoassay method using the hapten antigen-bound solid phase. It is to provide an immunoassay method using the solid phase.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the conventional problems, the present inventors have found that when binding a hapten antigen to a solid phase through an antigen-supporting substance, the binding ability to the solid phase is By simultaneously sensitizing a solid phase with a sensitizing substance that is 1/3 to 3 times that of the antigen-supporting substance, the influence of serum-derived components can be suppressed more than before, and
It was found that the hapten antigen or antibody in the sample to be measured can be measured more accurately than before, and an immunoassay method using the hapten antigen-bound solid phase was established to complete the present invention.

That is, in the present invention, an antigen-supporting substance to which a hapten antigen is bound and a sensitizing substance having a binding ability to the solid phase which is 1/3 to 3 times that of the antigen-supporting substance are simultaneously sensitized to the solid phase. by the hapten antigen and is bound antigen support material and the empathy work material is immobilized on the solid phase, then further provides a masking protein immobilized hapten antigen-binding solid phase comprising. The present invention also provides an immunoassay method performed using the hapten-bonded solid phase of the present invention.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The hapten antigen bound in the hapten antigen-binding solid phase of the present invention is not limited at all and may be any hapten antigen. Examples include, but are not limited to, steroidal hormones such as testosterone, progesterone and estradiol.

The hapten antigen is bound to an antigen support material. As the antigen supporting substance, a high molecular substance (molecular weight of 10,000 or more, preferably 50,000 to 1,000,000) is preferable,
Of these, proteins and lipids are preferable. Examples of preferred proteins include antibodies, bovine serum albumin (BS
A), casein, gelatin, ferritin, and the like, and examples of the lipid include, but are not limited to, gloposide-type glycolipids, ganglioside-type glycolipids, phospholipids, and the like. Among these, antibodies are particularly preferable, and as the antibodies, IgG, IgA, Ig
Antibodies derived from any animal such as E and IgM can be used. The antibody may be either a monoclonal antibody or a polyclonal antibody, but it must be an antibody having no reaction specificity to the hapten antigen to be measured. Since a large amount of the same molecule can be obtained with a monoclonal antibody, the hapten antigen-binding solid phase having specific characteristics can be reproducible by using both the antigen-supporting substance and the below-described sensitizing substance as a monoclonal antibody. There is an advantage that it can be manufactured well.

A covalent bond is preferred for binding the hapten antigen and the antigen-supporting substance, and known techniques can be appropriately used for the binding. For example, when a protein is used as the antigen supporting substance, the succinimide group introduced into the hapten antigen is reacted with the amino group of the protein or glycoprotein by the N-hydroxysuccinimide activated ester method to give the hapten antigen and the antigen supporting substance. Can be combined.

The reaction ratio between the hapten antigen and the antigen-supporting substance at this time affects the sensitivity of the measurement system, so the reaction conditions are set according to the desired sensitivity. Usually, the molar ratio of hapten antigen: antigen support material during the reaction is 0.1 to 100: 1.
However, in order to bind the hapten antigen and the antigen-supporting substance at a ratio of 1: 1, it is preferable to react them in approximately the same mole. Further, since the binding ratio of the antigen supporting substance and the hapten antigen is related to not only the molar ratio at the time of the binding reaction but also the reaction concentration at the binding reaction, the hapten antigen and the antigen supporting substance are bound at a target ratio. Therefore, it is necessary to pay attention to the reaction concentration. In general, when the reaction concentration is high, the hapten antigen-binding hapten antigen bound with a high ratio of hapten antigen can be prepared, and when the reaction concentration is low, the hapten antigen-binding hapten antigen with a low ratio of hapten antigen can be prepared.

In the present invention, the cosensitizing substance is sensitized at the same time as the antigen supporting substance to which the hapten antigen is bound. The cosensitizer has a binding ability to the solid phase of 1/3 of that of the antigen supporting substance.
It is 3 times, preferably 1/2 to 2 times, most preferably about 1 time. Here, the ability to bind to the solid phase is determined by sensitizing the solid phase with an equimolar mixture of the antigen supporting substance and the sensitizing substance.
It is defined by the molar ratio of the antigen-supporting substance and the cosensitizing substance immobilized on the solid phase. That is, for example, when the binding ability of the cosensitizer to the solid phase is three times that of the antigen-supporting substance,
When an equimolar mixture of a cosensitizer and an antigen-supporting substance is sensitized to a solid phase, it means that the number of moles of the cosensitizer immobilized on the solid phase is three times that of the antigen-supporting substance.

The binding ability of the sensitizing substance to the solid phase is 1/3 to 3 times that of the antigen-supporting substance (hereinafter, this may be referred to as "having equivalent binding ability"). The requirement can be easily achieved by adopting the same substance or a similar substance as the antigen supporting substance as the sensitizing substance. For example, when an antibody is used as the antigen supporting substance, the antibody may be used as the sensitizing substance. Antibodies have the same binding ability regardless of class, subclass, animal of origin, monoclonal antibody or polyclonal antibody, and the like. In particular, when the antigen-supporting substance and the co-sensitizing substance are both the same monoclonal antibody, the monoclonal antibody has completely the same binding ability to the solid phase since the antibody molecules are the same, and therefore, it has a certain property. This is advantageous because the hapten antigen-bound solid phase can be prepared with good reproducibility.

During the co-sensitization, the mixing ratio of the antigen-supporting substance to which the hapten antigen is bound and the co-sensitizing substance is 1: 1 in molar ratio.
˜1: 500 is preferable, and 1:10 to 1: 200 is more preferable. In order to reduce the amount of solid phase-bound hapten antigen to increase the sensitivity of the measurement system and to adjust the mixing ratio of the hapten antigen-binding antigen support material and the cosensitization material during cosensitization with good reproducibility, the hapten antigen is used. It is preferable to set the binding molar ratio of the antigen supporting substance to 1: 1 and to set the molar ratio of the antigen supporting substance to the cosensitizing substance at the time of cosensitization to about 1:10. In addition, particularly, when the hapten antigen molar ratio in the co-sensitization solution is set low, the mass of the sensitized crop during co-sensitization (that is,
It is preferable for preparing a hapten antigen-bound solid phase having high reproducibility that the total amount of the antigen-supporting substance to which the hapten antigen is bound and the sensitizing substance is approximately the same as the total adsorption capacity of the substance on the solid phase.

For the sensitization, generally used sensitization conditions can be appropriately used. For example, a co-sensitizing solution (that is, a mixed solution of an antigen-supporting substance to which a hapten antigen is bound and a co-sensitizing substance) and a solid phase are reacted at 37 ° C. for 1 hour to several hours, or at 4 ° C. overnight. Thus, the hapten antigen-binding solid phase can be prepared. In this case, the mass of the sensitized crop in the sensitizing solution is not particularly limited, but is 0.05 to
It is preferably about 10 mg, more preferably 0.1 to 3
It is about mg. Further, as the solvent for the co-sensitizing solution, a buffer solution usually used in this field can be used.

After the above-mentioned sensitization, usual masking with a masking protein such as BSA, skim milk, gelatin and animal serum is performed. The masking condition in this case is
The same as before, for example, a solution containing a masking protein in an amount of 0.1 to 20% (w / v) at 37 ° C.
The reaction may be performed for several hours to several hours or at 4 ° C. overnight. Non-specific adsorption can be further suppressed by masking.

The sensitized hapten antigen-bound solid phase of the present invention can be used in any of the known immunological measurement methods. When the measurement target is a hapten antigen, for example, a labeled hapten antigen-specific antibody, by a competitive reaction measurement method of competing the measurement target hapten antigen and the hapten antigen by the sensitization of the present invention, the measurement target hapten antigen When the target to be measured is an antibody, for example, the target to be measured is reacted with the hapten antigen-bound solid phase by the sensitization of the present invention, and the target to be measured is labeled anti-antibody. The amount of antibody can be detected. The antibody used in the immunoassay is not limited to a polyclonal antibody or a monoclonal antibody. Note that these immunoassay methods themselves are well known in this field, and are specifically described in the following examples.

[0018]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Reference Example 1 Preparation of Antibody-Binding Testosterone According to the method described in Japanese Patent Application Laid-Open No. 64-68661, succinimide of testosterone-3carboxymethoxime-N-hydroxysuccinimide ester and mouse anti-calcitonin monoclonal antibody KLG1-. The antibody-bound testosterone was prepared by reacting the amino group in lysine of 7 (home-made, hereinafter referred to as “KLG1-7”).

That is, 0.1M phosphate buffer pH
KLG1-7 prepared at a concentration of 10 mg / ml at 7.0
2.44 mg of dimethylformaldehyde in 1 ml
25 μl of testosterone-3carboxymethoxime-N-hydroxysuccinimide ester (manufactured by Boehringer Mannheim) adjusted to 1 ml / ml was added, and the mixture was allowed to stand at 30 ° C. for 1 hour to perform a coupling reaction. Then, the reaction solution was desalted with a PD-10 column Sephadex G-25M (manufactured by Pharmacia) equilibrated with 0.1 M phosphate buffer (pH 7.0) containing 0.1% sodium azide.

The concentration of the obtained testosterone-3carboxymethoxime-bound KLG1-7 solution (hereinafter referred to as testosterone-bound KLG1-7) was measured by measuring the absorbance.

Example 1 Preparation of particles by co-sensitization method of KLG1-7 and testosterone-bound KLG1-7 KLG1-7 and testosterone-bound KLG1-7 prepared in Reference Example 1 were added to 0.1M phosphate buffer pH 5.5. The mixture was dissolved and mixed to prepare KLG1-7 at 0.5 mg / ml and testosterone-bound KLG1-7 at 0.01 mg / ml, respectively, which were used as empathic buffer solutions. Then, magnetic particles (manufactured by Nippon Paint Co., Ltd.) 20 ultrasonically washed with distilled water 20
To the mg, 4 ml of the sympathetic action buffer was added, mixed well, and rotatably reacted at 37 ° C. for 1 hour with a rotator. After completion of the reaction, the magnetic particles were attracted with a magnet to collect them, the reaction solution was removed by suction, and ultrasonic cleaning with distilled water for 1 minute was performed 3 times. After washing, the sensitized magnetic particles were suspended in 50 mM female buffer solution pH 5.5, and 50 μl of 40 mg / ml 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (Nacalai Tesque, Inc.) aqueous solution was added. 2 with a rotator
Rotation reaction was performed at 5 ° C. for 30 minutes. The magnetic particles were collected by aspiration with a magnet, the reaction solution was removed by aspiration, and ultrasonic cleaning was performed for 1 minute with 50 mM bis-Tris buffer containing 150 mM sodium chloride and 0.1% sodium azide, pH 7.2.
I went there. Hereinafter, this particle is referred to as a synsensitized particle. After washing, the co-sensitized particles were suspended in 4 ml of the same buffer and stored at 4 ° C.

The resulting co-sensitized particles were ultrasonically washed three times for 1 minute with 50 mM bis-Tris buffer containing 150 mM sodium chloride, 0.1% sodium azide and 2% BSA, pH 7.2. The cells were suspended in 4 ml of the same buffer and stored at 4 ° C.

Comparative Example 1 Particle Preparation Method by Conventional Sensitization Method Testosterone-bound KLG1-7 prepared in Reference Example 1 was adjusted to 0.01 mg / ml with 0.1 M phosphate buffer p.
It was dissolved in H5.5 and used as a buffer for sensitization. The magnetic particles were sensitized in the same manner as in Example 1 (hereinafter referred to as conventional sensitized particles in the present specification). Conventional sensitized particles were stored at 4 ° C until use.

Comparative Example 2 Masking Treatment Conventional Sensitized Particles Conventional sensitized particles prepared in the same manner as in Comparative Example 1 were subjected to BSA masking treatment in the same manner as in Example 1 and used at 4 ° C. Saved in.

Example 2 Solid Phase Comparison Method and Results One Step Using Particles Before Masking Treatment in Example 1 and Four Kinds of Sensitized Particles Prepared in Example 1, Comparative Example 1 and Comparative Example 2 We compared non-specific reactions in competitive reactions. That is, each particle is treated with 5 particles containing BSA.
A particle solution was prepared by diluting 33.3 times with 0 mM Bis-Tris buffer pH 7.2 (hereinafter referred to as a particle diluent). On the other hand, the anti-testosterone antibody (manufactured by Dako) and the alkaline phosphatase derived from bovine small intestine (manufactured by Oriental Yeast Co., Ltd.) were used for the method described by Yoshitake et al., J. Biochem. 19
82, 92 (5), p1413-1424) to prepare an enzyme-labeled antibody, and a buffer for diluting the enzyme-labeled antibody contains 50 mM female buffer solution containing BSA, pH 6.8 (hereinafter, labeled antibody dilution). It is described as a liquid) and was 0.1 μg / ml. The standard antigen was prepared by adding a solution obtained by dissolving testosterone in ethanol to steroid-depleted serum (manufactured by AB T). As the samples, four non-specific samples showing inconsistency with the antibody solid phase method RIA (manufactured by Japan DPC) were used. As the substrate buffer and the washing solution, Lumipulse substrate solution (manufactured by Fujirebio) and Lumipulse washing solution (manufactured by Fujirebio) were used.

250 μl of particle solution, 50 μl of labeled antibody solution,
Using the standard antigen or 50 μl of the sample, 37 with the fully automated chemiluminescence immunoassay system Lumipulse (manufactured by Fujirebio)
One-step competition method measurement was performed at 30 ° C for 30 minutes. This operation was specifically performed as follows. That is, the particle liquid 250
50 μl of standard antigen or specimen and 50 μl of labeled antibody solution
Was added, and an immune reaction was carried out at 37 ° C. for 20 minutes, magnetic particles were separated from the reaction solution by magnetic force, and 200 μl of a cleaning solution was injected, stirred, and sucked repeatedly to perform cleaning. Then, 200 μl of the substrate solution was added and the enzyme reaction was carried out at 37 ° C. for 5 minutes, and then the luminescence amount was measured. The sample measurement value was calculated based on a calibration curve prepared from the luminescence amount of the standard antigen. All these operations were performed by computer control.

The results are shown in Table 1. The measured values obtained with the co-sensitized particles were lower than those of the conventional sensitized particles in all the cases, and were almost the same as those of the masked conventional particles. With the masking-treated co-sensitized particles, which are the particles of the present invention, the value was lower than the measured value of the co-sensitized particles, and the synergistic effect of the co-sensitization and the masking treatment was recognized.

[0029]

[Table 1]

Example 4 Examination of antibody co-sensitization concentration 1 Co-sensitized particles were prepared in the same manner as in Example 1 while changing the KLG1-7 concentration in the range of 0 to 2 mg / ml, and described in Example 3. Testosterone was measured in the same manner as in (1). As the specimens, 5 specimens that did not match with the antibody solid-phase method RIA (manufactured by Japan DPC) were used.

FIG. 1 shows the response (B / B ₀ (%)) of each standard antigen solution when the count value of the standard antigen 0 concentration is 100%.
The sample measured values are shown in FIG. B / B _{0 of} standard curve
(%) Was constant in any addition concentration range, and there was no change in sensitivity due to co-sensitization antibody concentration. The measured value of the sample rapidly increased to the antibody co-sensitization concentration of 0.2 mg / ml,
It gradually decreased from 0.2 to 0.5 mg / ml, and above that, it showed a constant measured value.

Example 5 Investigation of antibody co-sensitization concentration 2 According to the method described in Reference Example 1, progesterone-11α-carboxymethyl ester-N-hydroxysuccinimide ester (Boehringer Mannheim) KLG1- was used.
A progesterone-11α-carboxymethyl ester-bound KLG1-7 antibody bound to 7 (hereinafter referred to as progesterone-bound KLG) was prepared. This is KLG
Cosensitized particles were prepared in the same manner as in Example 1 while changing the 1-7 concentration in the range of 0 to 2 mg / ml, and the particles were suspended in a particle diluent.

On the other hand, the anti-progesterone monoclonal antibody (manufactured by Dako) and the alkaline phosphatase derived from bovine small intestine (manufactured by Oriental Yeast Co.) were used according to the method of Yoshitake et al.
Biochem. 1982) to prepare an enzyme-labeled antibody, which was adjusted to 0.1 µg / ml with an enzyme-labeled antibody diluent. The standard antigen was prepared by adding a solution obtained by dissolving progesterone in ethanol to steroid-depleted serum (manufactured by ABT Co.), and the measurement of progesterone was carried out in the same manner as in Example 3 except that the standard antigen and the sample amount were 20 μl. The procedure was as described. As the samples, 13 samples that did not match the antibody solid phase method RIA (manufactured by Japan DPC) were used.

The response (B / B ₀ (%)) of each standard antigen solution when the count value of the standard antigen 0 concentration is 100% is shown in FIG.
The measured values of the sample are shown in FIGS. B / B of standard curve
₀ (%) was constant in any addition concentration range, and there was no change in sensitivity due to the antibody concentration of co-sensitization. The measured value of the sample rapidly increased to the antibody co-sensitization concentration of 0.2 mg / ml,
It gradually decreased from 0.2 to 0.25 mg / ml, and above that, the measured value was constant.

Example 6 Examination of antibody co-sensitization concentration 3 In the same manner as the method described in Example 5, the antibody co-sensitization concentration was examined using the detection system for estradiol.

By the method described in Reference Example 1, estradiol-3-carboxypropyl ester N succinimide ester (manufactured by Boehringer Mannheim) was used as KLG1-7.
Estradiol-3-carboxypropyl ester-conjugated KLG1-7 antibody (hereinafter referred to as estradiol-conjugated KLG) was prepared. This is KLG
Cosensitized particles were prepared in the same manner as in Example 1 while changing the 1-7 concentration in the range of 0 to 0.5 mg / ml, and suspended in a particle diluent.

On the other hand, an anti-estradiol monoclonal antibody (manufactured by Interfarm) and alkaline phosphatase derived from bovine small intestine (manufactured by Oriental Yeast) were combined by the method of Yoshitake et al. (Supra) to prepare an enzyme-labeled antibody, which was then enzyme-labeled. The antibody dilution was adjusted to 0.1 μg / ml. The standard solution was prepared by adding a solution prepared by dissolving estradiol in ethanol to steroid-depleted serum (manufactured by ABT Co.), and the estradiol was measured by the method described in Example 3. The sample is the antibody solid-phase method RIA (Japan DP
Twelve cases of non-matching samples with C company) were used.

FIG. 6 shows the response (B / B ₀ (%)) of each standard antigen solution when the count value of the standard antigen 0 concentration is 100%.
7 and 8 show sample measured values. B / B of standard curve
₀ (%) was constant in any addition concentration range, and there was no change in sensitivity due to the antibody concentration of co-sensitization. The measured value of the sample rapidly decreased up to the antibody co-sensitization concentration of 0.125 mg / ml, and gradually decreased from 0.125 to 0.25 mg / ml, and showed a constant measured value above that.

[0039]

INDUSTRIAL APPLICABILITY According to the present invention, a hapten antigen-binding solid phase that is not affected by serum-derived components is provided, and an immunoassay method using the hapten antigen-binding solid phase provides a highly accurate immunoassay method. I was able to do it.

[Brief description of drawings]

FIG. 1 is a diagram showing a relationship between a concentration of a sensitizing substance and a standard solution B / B ₀ (%) using a testosterone measurement system.

FIG. 2 is a diagram showing a relationship between a concentration of a cosensitizer and a measured value of a discordant analyte using a testosterone measurement system.

FIG. 3 is a diagram showing a relationship between a concentration of a sensitizer and a standard solution B / B ₀ (%) using a progesterone measurement system.

FIG. 4 is a diagram showing a relationship between a concentration of a cosensitizer and a measured value of an inconsistent sample using a progesterone measurement system.

FIG. 5 is a diagram showing a relationship between a concentration of a cosensitizer and a measured value of a mismatched sample, using a progesterone measurement system.

FIG. 6 is a diagram showing a relationship between a concentration of a sensitizing substance and a standard solution B / B ₀ (%) using an estradiol measurement system.

FIG. 7 is a diagram showing a relationship between a concentration of a cosensitizer and a measured value of a mismatched sample using an estradiol measurement system.

FIG. 8 is a diagram showing a relationship between a concentration of a co-sensitizer and a measured value of a mismatched sample using an estradiol measurement system.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI G01N 33/543 565 G01N 33/543 565W 33/577 33/577 B (72) Inventor Isao Nishizono 2 Nihonbashihamacho, Chuo-ku, Tokyo Inc. 62-5, Fujirebio Co., Ltd. (72) Inventor Yoshihiro Koyamaishi 2-62-5, Nihonbashihamacho, Chuo-ku, Tokyo In Fujirebio Co., Ltd. (56) Reference JP-A-7-107992 (JP, A) JP-A-8-231591 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 33/53 G01N 33/543 G01N 33/577

Claims (12)

(57) [Claims] 1. A solid phase is simultaneously sensitized with an antigen supporting substance to which a hapten antigen is bound and a sensitizing substance having a binding ability to the solid phase which is 1/3 to 3 times that of the antigen supporting substance. the hapten antigens is bound to the antigen support material and the sympathy work material is immobilized on the solid phase, then further masking protein immobilized by comprising hapten antigen-binding solid phase. 2. The hapten antigen-binding solid phase according to claim 1, wherein the antigen-supporting substance and the sensitizing substance are proteins. 3. The hapten antigen-binding solid phase according to claim 2, wherein the protein is an antibody. 4. The hapten antigen-binding solid phase according to any one of claims 1 to 3, wherein the antigen-supporting substance and the sensitizing substance are the same substance. 5. The hapten antigen-binding solid phase according to claim 4, wherein the antigen-supporting substance and the sensitizing substance are the same monoclonal antibody. 6. The molar ratio of the antigen-supporting substance to which the hapten antigen bound for sensitization is bound to the co-sensitizing substance is 1: 1.
The hapten antigen-binding solid phase according to any one of claims 1 to 5, wherein the solid phase is ˜1: 500. 7. The hapten antigen-binding solid phase according to claim 6, wherein the molar ratio is 1:10 to 1: 200. 8. The hapten antigen-binding solid phase according to claim 1, wherein the masking protein is bovine serum albumin. 9. The hapten antigen-binding solid phase according to claim 1, wherein the hapten antigen is a steroid hormone. 10. The hapten antigen-binding solid phase according to claim 9, wherein the steroid hormone is testosterone, progesterone or estradiol. 11. An immunoassay method performed by using the hapten antigen-bound solid phase according to claim 1. 12. The method according to claim 11, wherein the hapten antigen in the sample is measured by utilizing a competitive reaction between the hapten antigen bound to the solid phase and the hapten antigen in the sample.