JP2002097197A - Estradiol derivative and assaying method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a high-sensitive immunoassay for estrogens. SOLUTION: This method is a high-sensitivity immunoassay for estrogens, comprising using as the marker a biotinated estradiol derivative of the general formula (1) (wherein, R1 and R2 are such that one of them H, the other being a group of the general formula (2); in the group, R3 is an arginine residue or lysine residue; x is an integer of 0 or 1; y is an integer of 1-5; and z is an integer of 1-3).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an estrogen (est).
rogens), in particular, an immunoassay for 17β-estradiol and estrone and a label used in the assay.

[0002]

2. Description of the Related Art Estrogen is a typical example of a steroid hormone (female hormone). At present, dozens of compounds belonging to estrogen are known. This estrogen is metabolized to stable estradiol in human blood like other female hormones. This estradiol is mainly excreted as urine and is diffused into the environment such as rivers, lakes, seawater, drainage and soil.

[0003] Estrogen attracts attention in terms of feminization of humans, livestock, fish and the like. For example, 17-estradiol reacts with the estrogen receptor. The binding activity of estradiol to the receptor is as much as 10,000 to 10,000 times higher than that of bisphenol A, nonylphenol, alkyl phthalates, etc., which are currently considered environmental hormones.

Conventionally, 17β-estradiol and estrone have been measured by immunoassays such as radioimmunoassay and enzyme immunoassay. These immunoassays are known as measurement techniques for measuring female hormones in human blood.

However, these measuring methods are originally
It was developed for the detection and measurement of female estrogen in blood.When used for the detection and measurement of estrogen in environments such as rivers, lakes, seawater, drainage and soil, there are still issues in terms of sensitivity and other issues. Have left.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a highly sensitive estrogen immunoassay system which solves the problems of the prior art.

[0007]

Means for Solving the Problems As a result of intensive studies, the present inventor has found that when a novel biotinylated estradiol derivative represented by the following general formula (1) is used as a label in an estrogen immunoassay system, the present inventors have found that We found that a sensitive estrogen detection and measurement system could be established. The present invention has been completed based on this finding.

The present invention provides a compound represented by the general formula (1)

[0009]

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Wherein ^one of R ¹ and R ² is a hydrogen atom,
The other is based

[0011]

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FIG. In the group, R ³ is the same or different and arginine residue or lysine residue, x is 0 or
1, y represents an integer of 1-5 and z represents an integer of 1-3. Biotinylated estradiol derivatives represented by:
(Hereinafter, referred to as compound (1)).

Particularly, the present invention relates to a compound represented by the formula (1)¹Is a hydrogen source
Child, R^ThreeIs an arginine residue, X is 1, Y is 2 and Z is 2.
Biotinylated estradiol derivative, and R^TwoIs a hydrogen source
Child, R ^ThreeIs an arginine residue, X is 0, Y is 1 and Z is 2.
A biotinylated estradiol derivative is provided.

The present invention also provides an immunoassay for estrogen, particularly an immunoassay for 17β-estradiol and estrone, which comprises using compound (1) as a label in an assay system.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The compound (1) of the present invention is characterized by being soluble (soluble) in the liquid phase of an immunoassay system. Therefore, this is extremely useful as a label in an estrogen immunoassay system.

Heretofore, biotin has been commonly used as an indirect labeling agent together with avidin. In addition, biotin is considered to be more preferably used as a label because it has a low molecular weight (molecular weight 244.31) as compared with general enzymes. However, biotin itself is poor in water solubility, and the conjugate obtained by binding to estradiol which lacks water solubility becomes water-insoluble. Therefore, biotin cannot be used in such an estrogen immunoassay system.

On the other hand, the compound (1) of the present invention has water solubility suitable for use in an immunoassay system for estrogen. Moreover, the compound (1) has not lost its cross-reactivity with the antibody used in the estrogen measurement system.
From these points, the compound (1) is suitable for use in an immunoassay system for estrogen, which is required in the art.

One preferred embodiment of the assay of the present invention is the enzyme immunoassay described in detail in Example 3 below. According to this preferred embodiment, 17β-estradiol in the range of about 1.2-5000 pg / mL can be measured. Also, about 1.2-20000p
Estrone in the g / mL range can be measured.

From these facts, the measuring method of the present invention is a very sensitive measuring method which can measure estrogen existing in these environments using, for example, rivers and lakes as samples.

The compound (1) of the present invention can be advantageously used as a label in an immunoassay for estrogen, particularly 17β-estradiol and estrone, which are known to have high physiological activity.

In the above general formula (1) representing the compound (1), the amino acid residues (Arg and Lys) represented by R ³ include both D-form and L-form unless otherwise specified.

The compound (1) of the present invention has, for example, the following general formula (2)
(Hereinafter referred to as compound (2)) and a compound represented by the general formula
It can be produced by a condensation reaction with the compound represented by (3) (hereinafter, referred to as compound (3)).

[0023]

Embedded image

(Wherein one of R ⁴ and R ⁵ is a hydrogen atom,
The other is the group - shows the _{(CH 2) y -COOH - (} CO) x. R ³ , x, y and z
Is the same as above. Compound (3) can be prepared by, for example, biotin hydrazide and one type of desired amino acid (z = 1
) Or two or more (when z = 2 or 3) are sequentially subjected to a condensation reaction. The condensation reaction can be performed according to various known condensation reaction methods. This condensation reaction method includes a usual liquid phase method and a solid phase method.

As the condensation reaction method, for example, an azide method,
Mixed acid anhydride method, DCC method, active ester method, redox method, DPPA (diphenylphosphoryl azide) method, DCC + additive method (additives: 1-hydroxybenzotriazole, N-hydroxysuccinimide, N-hydroxy-5- Norbornene-
2,3-dicarboximide) and the Woodward method.

The solvent used in each of these methods can be appropriately selected from various solvents commonly used in this kind of peptide condensation reaction. Examples thereof include N-methylpyrrolidone (NMP), dimethylformamide (DMF), dimethylsulfoxide (DMSO) and the like, and a mixed solvent thereof.

In the above condensation reaction, functional groups which do not participate in the reaction can be protected by a usual protecting group according to a conventional method, and these can be eliminated after completion of the reaction. These respective reaction methods are known, and the reagents used for them can be appropriately selected from known ones.

For example, amino-protecting groups include benzyloxycarbonyl (Z), tert-butoxycarbonyl
(Boc), isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl and the like.

Examples of the carboxyl-protecting group include groups capable of forming lower alkyl esters such as methyl ester, ethyl ester and tertiary butyl ester, benzyl ester and p-methoxybenzyl ester.

The protective group for the guanidino group of arginine includes, for example, 2,2,5,7,8-pentamethylchromone-6-sulfonyl (Pmc), 2,2,4,6,7-pentamethyldihydrobenzofuran- Examples include 5-sulfonyl (Pbf), nitro, Z, Boc, p-toluenesulfonyl and the like.

The elimination reaction of these protecting groups is also carried out by a commonly used method, for example, catalytic reduction method, liquid ammonia / sodium, hydrogen fluoride, hydrogen bromide, hydrogen chloride, trifluoroacetic acid (TFA), acetic acid, methanesulfonic acid and the like. Can be carried out in accordance with a method using

The hydrazide of biotin is, for example, Z, Boc,
Manufacture by subjecting a hydrazine protected with an optional protecting group such as Troc (N-trichloroethoxycarbonyl group) to biotin to a condensation reaction in the same manner as described above, and then removing the protecting group of the resulting compound. Can be.

The condensation reaction between the compound (3) and the compound (2) can be carried out according to the above-mentioned condensation reaction, and the introduction and elimination of the protecting group can be carried out in the same manner.

The obtained compound (1) can be appropriately purified by various purification means such as high performance liquid chromatography according to a conventional method.

According to the use of the compound (1), estrogens, particularly, estrogen, can be obtained by various immunoassays such as enzyme immunoassay (EIA), enzyme immunometric assay (ELISA), fluorescence immunoassay and luminescence immunoassay. Highly sensitive detection and measurement of 17β-estradiol or estrone is possible.

The immunoassay of the present invention essentially requires the use of the compound (1) of the present invention as a label in an assay system, and other than that, various immunoassays generally used, for example, various methods such as a competition method and a sandwich method are used. It can be performed according to the method.

The antibody of the measurement system can be used by immobilizing it on any desired solid phase, if desired. As the solid phase, various solid phases commonly used in this kind of technical field can be used. Immobilization of the antibody on the solid phase is also not particularly limited, and can be performed by any of ordinary physical bonding and chemical bonding.

The immunological reaction in the immunoassay according to the present invention is not particularly limited, either.
For example, the reaction can be carried out generally at a temperature of 45 ° C. or lower, usually about 4-40 ° C., for about 0.5 to several hours.

The solvent used in the reaction and the pH of the reaction system are not particularly limited as long as they do not adversely affect the reaction. Examples of the solvent include, for example, citrate buffer, phosphate buffer, Tris salt buffer, acetate buffer and the like.

The method of the present invention is preferably carried out using the compound of the present invention.
According to the competition method using (1) as a label, it can be carried out as follows. For example, first, a desired antibody (anti-estrogen antibody) directed to an estrogen to be measured is immobilized on an immobilized antibody in the presence of the compound (1) of the present invention as a label. An antigen-antibody reaction (competition) is performed by adding a sample that may contain estrogen. Next, the label bound to the immobilized antibody is measured using a detection reagent comprising ordinary avidin.

The detection reagent comprising avidin is not particularly limited, and for example, avidin or streptavidin modified with various labeling agents generally used in the art can be used.

The labeling agent is not particularly limited, and any conventionally known labeling agent can be used. Preferred specific examples include enzymes such as alkaline phosphatase (ALP) and peroxidase (HRP). Modification of avidin with these labeling agents can be performed according to a known method. These modified products are also available as commercial products.

The immunoassay of the present invention is particularly preferably
It can be carried out according to a high-sensitivity ELISA described in Examples described later.

One preferred embodiment of the immunoassay of the present invention includes an immunoassay / detection method for 17β-estradiol. In this method, preferably, the compound (1) of the present invention in which R ² is a hydrogen atom is used as a label in the measurement system. In addition, an antibody reactive with 17β-estradiol is used as a specific antibody in the measurement system. The antibody can be obtained, for example, by using a compound (2) in which R ⁵ is a hydrogen atom as an immunogen according to the method described below.

Another preferred embodiment of the immunoassay of the present invention includes an immunoassay for estrone. In this method, preferably, the compound (1) of the present invention in which R ¹ is a hydrogen atom is used as a label in the measurement system. Further, an antibody having reactivity with estrone is used as a specific antibody in the measurement system. The antibody can be obtained, for example, by using a compound (2) in which R ⁴ is a hydrogen atom as an immunogen according to the method described below.

The estrogen immunoassay according to the present invention can be carried out in consideration of the simplicity of its operation and the like.
It is preferably carried out using a measurement kit containing (1) as an active ingredient. The measurement kit can further contain other reagents necessary for performing the detection and measurement, such as the above-mentioned detection reagent composed of avidin, an anti-estrogen antibody, a standard antigen, and an assay buffer.

The immunoassay of the present invention can be used for rivers, lakes, seawater,
It is particularly useful for detecting and measuring estrogen in these environments, using wastewater, soil and the like as test substances. The present invention is also useful for detecting and measuring estrogens in human and animal tissues, blood, urine, bone marrow fluid, etc. and fish tissues, blood, etc. as test substances.

The anti-estrogen antibody used in the immunoassay of the present invention is not particularly limited as long as it has specific reactivity to the estrogen to be measured. This antibody includes the above-mentioned antibodies or antibodies produced according to them. Examples thereof include antisera of warm-blooded animals, polyclonal antibodies such as chicken egg antibodies, and monoclonal antibodies.

These antibodies can be appropriately selected and used depending on the estrogen to be measured. Preferred examples of antibodies include antibodies reactive with 17β-estradiol and / or estrone.

It has been confirmed by the present inventors that polyclonal antibodies are generally superior to monoclonal antibodies in titer of an antibody produced using a low-molecular organic compound such as estrogen as an antigen. Therefore, it is preferable to use a polyclonal antibody also in the immunoassay of the present invention.

[0051] Polyclonal antibodies are produced in animals by immunizing warm-blooded animals (excluding humans) with immunizing antigens. The immunization method or technique can basically follow a conventional method.

As the immunizing antigen, preferably compound (2)
And a carrier protein can be used.

As the carrier protein, various carrier proteins known in the art to enhance the immunogenicity of the antigen or hapten can be used without limitation. Examples thereof include various animal proteins such as albumin, globulin, thioglobulin, and hemocyanin, and polylysine.

The binding reaction between the carrier protein and the compound (2) is carried out in the same manner as the above-mentioned condensation reaction.
The reagent used at that time may be a commonly used reagent.

The carrier protein conjugate of compound (2) itself has desired immunogenicity and can be used as an immunogen for producing a specific antibody which can be preferably used in the assay method of the present invention.

As the immunizing antigen, an adsorbent obtained by further adsorbing the carrier protein conjugate on a suitable polymer adsorbent can also be used.

As the polymer adsorbent, various polymer substances known in the art to enhance immunogenicity can be used. Examples thereof include serum proteins such as polyvinylpyrrolidone, latex, butathioglobulin, and carbon powder.

The desired adsorbent can be obtained by mixing these polymer adsorbents with the carrier protein conjugate according to a conventional method.

The immunization and the operation of obtaining a desired antibody can be performed according to a conventional method. For example, a warm-blooded animal such as a rabbit, sheep, guinea pig, or chicken is injected multiple times with an emulsion prepared by mixing the above antigen with Freund's complete adjuvant, and the obtained antiserum is obtained according to a conventional method. By doing so, a polyclonal antibody can be obtained. When a chicken is used, the above antigen is immunized multiple times, and immunoglobulin is added to the chicken eggs laid by the chicken.
The desired polyclonal antibody can also be obtained by producing (IgY) and obtaining IgY from the egg yolk of the chicken egg according to a conventional method.

The specific antibody that can be used in the assay of the present invention may be a monoclonal antibody. This monoclonal antibody is produced, for example, by immunizing a mouse with the above antigen multiple times together with Freund's complete adjuvant to immunize the mouse, and then producing the antibody-producing cells and bone marrow cells or spleen cells by conventional methods such as cell fusion. Fused according to law,
It can be obtained by cloning, isolating a single clone cell producing the desired antibody, and culturing the cell.

Each antibody thus obtained may be optionally used
It can be further purified according to a conventional method such as ammonium sulfate salting out, ion exchange chromatography, affinity chromatography and the like.

[0062]

The present invention will be described in more detail with reference to the following examples. These examples are non-limiting and the scope of the invention should not be construed as being limited by these examples.

Example 1 Production of Antibody (1) β-Estradiol 3-carboxymethyl ether (6.3 mg, 0.019 mmol; Sigma) was added to 100 μL of DMSO and 0.1 MP
Keyhole limpet hemocyanin (KLH; 20 mg; Pierce C) dissolved in 1 mL of BS (pH 5.0) and dissolved in 1 mL of the same buffer
Chemical Company) and water-soluble carbodiimide (10m
g; Peptide Research Laboratories), water-soluble carbodiimide (178 mg) was added, and the mixture was reacted at room temperature for 30 minutes.

The above reaction solution (2 mL) was mixed with 50% polyvinylpyrrolidone (2 mL; Merck), and further emulsified by adding an equal amount of Freund's adjuvant (4 mL; Calbiochem).

The resulting emulsion was intradermally injected into three rabbits (Japanese white, male, weighing 2.0-2.5 kg) so that the immunizing antigen was 2 mg / rabbit. Booster immunizations were performed by repeating the injection immunization six times every two weeks at a dose of half the initial injection volume. One week after the final immunization, whole rabbit blood was collected after the production of a sufficient titer of antiserum was confirmed. Leave the blood at 37 ° C for 1 hour, then at 4 ° C overnight,
An antiserum was obtained by centrifugation at 3000 rpm, which was freeze-dried.

(2) An emulsion was prepared in the same manner as in (1) above, using β-estradiol-17-succinate (6.3 mg; Sigma). The obtained emulsion was prepared as an immunizing antigen for 3 rabbits at 1 mg / rabbit and injected intradermally,
An antiserum was obtained in the same manner as in the above (1).

[0067]Example 2 Biotinylated estradiol induction
Preparation of conductor (1) Boc-Arg (Z)_Two-NHNH-biotin (protecting group Z: benzyloxy
Synthesis of water-soluble carbodiimide (212 μL, 1.2 mmol) with Boc-Arg
(Z)_Two-OH (543 mg, 1.0 mmol), HOBt (162 mg, 1.2 mmol) and
Biotin-N_TwoH_Three(258 mg, 1.0 mmol) in DMF solution (10 mL)
After dropwise addition at 0 ° C. and stirring at room temperature for 18 hours, the solvent was distilled off.
Distilled water was added to the residue under ice cooling to solidify, and the precipitate was collected.
Recrystallized from DMF-ethyl acetate, Boc-Arg (Z) _Two-CONHNH-
Biotin was obtained at 775 mg (99.1%).

(2) Synthesis of Boc-Arg (Z) ₂ -Arg (Z) ₂ -NHNH-biotin Boc-Arg (Z) ₂ -NHNH-biotin prepared in (1) above (352 mg,
(0.45 mmol) was dissolved in TFA (3 mL), and the mixture was stirred under ice-cooling for 30 minutes, and treated with ether to remove Boc. The Boc-Arg (Z) ₂ -OH (293 mg, 0.5
4mmol) and water-soluble carbodiimide (114μL, 0.65mmol) / HO
A condensation reaction was performed in DMF (5 mL) using Bt (88 mg, 0.65 mmol), the solvent was distilled off, and the residue was solidified by adding distilled water under ice-cooling, and recrystallized from DMF-ethyl acetate. 502 mg (92.4%) of the desired product was obtained.

(3) Biotinylated estradiol derivative
1: Compound (1) (R ¹ = hydrogen atom, R ³ = arginine residue, x = 1,
Synthesis of y = 2 and z = 2) Boc-Arg (Z) ₂ -Arg (Z) ₂ -NHNH-biotin (65.
2 mg, 0.054 mmol) was treated with TFA (0.5 mL) for 30 minutes under ice-cooling to remove boc. The obtained de-Boc form, β-estradiol-17-succinate (20.1 mg, 0.054 mmol)
And HOBt (8.8 mg, 0.065 mmol) in DMF (2 mL), and then dissolved in water-soluble carbodiimide (11.4 μL, 0.065
mmol), and the mixture was stirred at room temperature for 18 hours.
Distilled water was added to the residue to solidify it. Reprecipitation from DMF-ethyl acetate gave 60 mg of the desired compound (Arg protected form).

This protected product was treated with methanol (10 mL) -50% acetic acid (1%).
(0 mL) in the presence of 10% Pd-C (10 mg) in a mixed solvent, and was subjected to catalytic reduction at room temperature for 4 hours by passing hydrogen gas. After removing the catalyst by filtration, the filtrate was freeze-dried to obtain 40 mg of a crude product.

After dissolving the crude product (40 mg) in 50% acetic acid (2 mL),
Purified by reverse-phase preparative HPLC using a column (YMC-PackD-ODS-5; 20 × 250 mm) using 1% TFA / CH ₃ CN mixed solvent system (75/25 → 60/40, 30 minutes) as an eluent. 26 mg of the desired title compound
Obtained.

Fab-mass spectrum: theoretical value (C ₄₄ H ₆₈ N ₁₂ O ₈ S + H) ⁺ : 925 actual value: 925.168 (4) biotinylated estradiol derivative 2: compound (1) (R
² = hydrogen atom, R ³ = arginine residue, x = 0, y = 1 and z = 2)
Synthesis of Target Biotinylated estradiol derivative 2 was synthesized in the same manner as in (3) above. That is, Boc-Arg (Z) ₂ -Arg
After treating (Z) ₂ -NHNH-biotin (20.4 mg, 0.019 mmol) with TFA,
β-estradiol-3-carboxymethyl ether (6.
3mg, 0.019mmol) and DMF (1mL) in water-soluble carbodiimide /
Condensed by the HOBt method. The same post-treatment was performed to obtain 21 mg of the protected compound of the target compound. Methanol (5 mL)-
Catalytic reduction through hydrogen gas in the presence of 10% Pd-C (3 mg) in a mixed solvent of 50% acetic acid (5 mL) gave 14 mg of a crude product. This crude product (14 mg) was dissolved in 50% acetic acid (1.5 mL), and purified in the same manner by preparative reverse-phase HPLC to obtain 3.5 mg of the desired compound.

Fab-mass spectrum: theoretical value (C ₄₂ H ₆₆ N ₁₂ O ₇ S + H) ⁺ : 883 actual value: 883.133 Example 3 Immunoassay system (1) 17β-estradiol using the derivative of the present invention as a label The ELISA system for was set up as follows.

(1-1) Reagents and materials: <Assay buffer> PBS (-) containing 0.1% BSA <96-well plate> Goat anti-rabbit IgG (Fc) antibody-immobilized 96-well plate <Labeled body> Biotinylated estradiol derivative 2 <HRP labeling reagent> Streptavidin HRP (Calbiochem) <Substrate> TMB (Tetramethyl benzidine) solution (Sigma) <Antiserum> RY725 (rabbit antiserum obtained from (1) of Example 1) <Standard product> 17β-estradiol (Sigma) <Washing solution> Physiological saline containing 0.05% Tween 20 <Enzyme reaction stop solution> 2N sulfuric acid (1-2) ELISA procedure: (a) Standard for each well of a 96-well plate Liquid or specimen 10
After dispensing 0 μL, 50 μL of a labeled substance (0.2 ng / mL) composed of the biotinylated estradiol derivative of the present invention and antiserum (×
32000) Add 50 μL.

(B) React at 4 ° C. overnight (18-20 hours).

(C) Wash all wells four times with a washing solution.

(D) The reaction is carried out at room temperature for 2 hours using a microplate shaker.

(E) Wash all wells four times with a washing solution.

(F) Add 100 μL of substrate.

(G) React at room temperature for 20 minutes.

(H) Dispense 100 μL of the enzyme reaction stop solution into each well.

(I) Using a microplate auto reader
Measure absorbance at 450 nm.

(1-3) Results A standard curve of 17β-estradiol ELISA is shown in FIG.

From the results shown in the figure, it can be seen that 17β-
The estradiol measurement range was 1.2-5000 pg / mL.

(2) An ELISA system for estrone using the derivative of the present invention as a label was set up as follows.

(2-1) Reagents and materials: <Assay buffer> PBS (-) containing 0.1% BSA <96-well plate> Goat anti-rabbit IgG (Fc) antibody-immobilized 96-well plate <Labeled body> Biotinylated estradiol derivative 1 <HRP labeling reagent> Streptavidin HRP (Calbiochem) <Substrate> TMB (Tetramethyl benzidine) solution (Sigma) <Antiserum> RY469 (rabbit antiserum obtained from (2) of Example 1) ) <Standard product> Estrone (Sigma) <Washing solution> Physiological saline containing 0.05% Tween 20 <Enzyme reaction stopping solution> 2N sulfuric acid (2-2) ELISA procedure: (a) Standard solution is added to each well of a 96-well plate. Sample 10
After dispensing 0 μL, 50 μL of a label (0.1 ng / mL) composed of the biotinylated estradiol derivative of the present invention and antiserum (×
32000) Add 50 μL.

(B) Reaction is performed for 5 hours using a room-temperature microplate shaker.

(C) Wash all wells four times with a washing solution.

(D) Add 100 μL of HRP labeling reagent (× 5000).

(E) The reaction is carried out at room temperature for 1 hour using a microplate shaker.

(F) Wash all wells four times with a washing solution.

(G) Add 100 μL of substrate.

(H) React at room temperature for 20 minutes.

(I) Dispense 100 μL of the enzyme reaction stop solution into each well.

(J) Using a microplate auto reader
Measure absorbance at 450 nm.

(2-3) Results FIG. 2 shows a standard curve of estrone ELISA.

From the results shown in FIG. 2, the estrone measurement range of this measurement system was 1.2-20000 pg / mL.

Comparative Example 1 (1) In Example 1 (1), a commercially available labeled substance was used in place of the derivative of the present invention as the labeled substance in the measurement system.
An ELISA system for β-estradiol was set up.

(1-1) Reagents and materials: <Assay buffer> PBS (-) containing 0.1% BSA <96-well plate> Goat anti-rabbit IgG (Fc) antibody-immobilized 96-well plate <Labeled substance> Estradiol HRP Labeling reagent (Estradiol-3-peroxidas
e labelled; Cosmo) <Substrate> TMB (Tetramethyl benzidine) solution (Sigma) <Antiserum> RY725 (rabbit antiserum obtained in (1) of Example 1) <Standard> 17β-estradiol (Sigma) <Washing solution> Physiological saline containing 0.05% Tween 20 <Enzyme reaction stop solution> 2N sulfuric acid (1-2) ELISA procedure: (a) Standard solution or sample 10 in each well of a 96-well plate
After dispensing 0 μL, 50 μL of a label (× 100) composed of an estradiol HRP labeling reagent and 50 μL of antiserum (× 8000) are added.

(B) React overnight at 4 ° C.

(C) Wash all wells four times with a washing solution.

(D) Add 100 μL of substrate.

(E) React at room temperature for 30 minutes.

(F) Dispense 100 μL of the enzyme reaction stop solution into each well.

(G) Using a microplate auto reader
Measure absorbance at 450 nm.

(1-3) Results A standard curve of 17β-estradiol ELISA is shown in FIG.

From the results shown in FIG. 3, the 17β-
The estradiol measurement range was 19.5-5000 pg / mL.

(2) The following ELISA system for estrone was set up in (2) of Example 3 using a commercially available label instead of the derivative of the present invention as the label in the measurement system.

(2-1) Reagents and materials: <Assay buffer> PBS (-) containing 0.1% BSA <96-well plate> Goat anti-rabbit IgG (Fc) antibody-immobilized 96-well plate <Labeled substance> Estradiol HRP Labeling reagent (Estradiol-17-peroxidas
e labeled; Sigma) <Substrate> TMB (Tetramethyl benzidine) solution (Sigma) <Antiserum> RY469 (rabbit antiserum obtained in (2) of Example 1) <Standard product> Estrone (Sigma) <Washing solution > Physiological saline containing 0.05% Tween 20 <Enzyme reaction stop solution> 2N sulfuric acid (2-2) ELISA procedure: (a) Standard solution or sample 10 in each well of a 96-well plate
After dispensing 0 μL, 50 μL of a label (0.25 μg / mL) composed of an estradiol HRP labeling reagent and 50 of antiserum (× 32000)
Add μL.

(B) Reaction is performed for 5 hours using a room-temperature microplate shaker.

(C) Wash all wells four times with a washing solution.

(D) Add 100 μL of substrate.

(E) React at room temperature for 20 minutes.

(F) Dispense 100 μL of the enzyme reaction stop solution into each well.

(G) Using a microplate autoreader
Measure absorbance at 450 nm.

(2-3) Results FIG. 4 shows a standard curve of estrone ELISA.

From the results shown in FIG. 4, the estrone measurement range of this measurement system was 4.8-20000 pg / mL.

Example 4 17β-estradiol ELISA System
Cross-reactivity test of Example 3 In (1) of Example 3, in place of 17β-estradiol (Sigma) used as a standard, each estrogen shown in Table 1 below was used, and ELISA was repeated in the same manner. A curve was created (see Figure 5).

Using the obtained standard curve of each estrogen and the standard curve of 17β-estradiol prepared in (1) of Example 3, the reactivity between 17β-estradiol and antiserum in this measurement system was determined. The reactivity between each estrogen and the antiserum (cross-reactivity with 17β-estradiol) was determined when the ratio was set to 100%. The results are shown in Table 1 below.

[0120]

【table 1】

Example 5 Cross-reactivity of the estrone ELISA system
In Test Example 3 (2), ELISA was repeated in the same manner using each estrogen shown in Table 1 below in place of estrone (Sigma) used as a standard, to prepare a standard curve (FIG. 6).

Using the obtained standard curve of each estrogen and the standard curve of estrone prepared in (2) of Example 3, the reactivity between estrone and antiserum in this measurement system was set to 100%. At this time, the reactivity between each estrogen and the antiserum (cross-reactivity with estrone) was determined. The results are shown in Table 2 below.

[0123]

[Table 2]

[Brief description of the drawings]

FIG. 1 is a standard curve in the immunoassay of the present invention prepared according to Example 3 (1).

FIG. 2 is a standard curve in the immunoassay of the present invention prepared according to (2) of Example 2.

FIG. 3 is a standard curve in a comparative immunoassay prepared according to (1) of Comparative Example 1.

FIG. 4 is a standard curve in a comparative immunoassay prepared according to (2) of Comparative Example 1.

FIG. 5 is a graph showing the reactivity of each estrogen with an antiserum in a cross-reactivity test according to Example 4.

FIG. 6 is a graph showing the reactivity of each estrogen with an antiserum in a cross-reactivity test according to Example 5.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Kazuyuki Kitamura 3289-13 Manohara Nitta, Fujinomiya-shi, Shizuoka Prefecture F-Meal C201 (72) Inventor Tsukasa Kodaira 56-7F, Minamikawako, Hiroshima, Matsushimo-cho, Itano-gun, Tokushima Prefecture F Terms (reference) 4C091 AA01 BB03 BB04 BB07 CC03 DD01 EE04 EE05 FF01 GG01 HH01 JJ01 KK01 LL01 MM03 NN01 PA09 QQ01

Claims (6)

[Claims] [Claim 1] General formula (1): (Wherein ^one of R ¹ and R ² is a hydrogen atom and the other is a group Is shown. In this group, R ³ is the same or different and represents an arginine residue or a lysine residue, x is 0 or 1, y is an integer of 1-5, and z is an integer of 1-3. ) A biotinylated estradiol derivative represented by the formula: 2. R ¹ is a hydrogen atom, R ³ is an arginine residue, and X is
2. The biotinylated estradiol derivative according to claim 1, wherein 1, Y is 2 and Z is 2. 3. R ² is a hydrogen atom, R ³ is an arginine residue, and X is
2. The biotinylated estradiol derivative according to claim 1, wherein 0, Y is 1 and Z is 2. 4. An immunoassay for estrogen, comprising using the biotinylated estradiol derivative according to claim 1 as a label in a measurement system. 5. The immunoassay according to claim 4, wherein the estrogen is 17β-estradiol. 6. The method according to claim 4, wherein the estrogen is estrone.
The immunoassay according to 4.