JPH0242194B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antigen for producing an antibody for immunochemical measurement comprising _three types of vitamin D.

More specifically, the present invention provides novel vitamins having a carboxyl group or an ester group thereof at the 22nd position.
The present invention relates to an antigen for producing antibodies for enzyme immunoassay or radioimmunoassay consisting of a D ₃ derivative and an immunogenic carrier.

Vitamin D ₃ is metabolized in the kidney or liver and converted into 25-hydroxyvitamin D ₃ ,
It is known that it is converted into 1α,25-dihydroxyvitamin _D3 , 24,25-dihydroxyvitamin _D3, etc. And these metabolites such as 25-hydroxyvitamin D ₃ , 1α, 25-dihydroxyvitamin D ₃ or 1α-hydroxyvitamin D ₃ ,
Active vitamin D ₃ compounds such as 1α,24-dihydroxyvitamin D ₃ are used clinically as therapeutic agents for osteoporosis, osteomalacia, etc., or are under development. Clinical doses of these drugs are usually significantly lower, with a single dose of several hundred nanograms.
It is in the range of several tens of micrograms.

On the other hand, pharmacological action generally has a correlation with the serum concentration or tissue concentration of a drug, and the measurement of the serum concentration of a drug in humans is of particular clinical importance.

Conventionally, a radioreceptor assay method using 1α,25-dihydroxyvitamin _D3 receptors has been known as a method for quantifying active vitamin _D3 compounds (Biochem-
istry), 13, 4091 (1974)). This assay method requires a lot of time and effort to carry out, requires a large amount of serum, and has the drawbacks that its accuracy is not fully satisfactory.

As another assay method, 1α-hydroxyvitamin D ₃ 3-hexuccinate or 1α,25-dihydroxyvitamin is disclosed in JP-A-55-47653.
A radioimmunoassay method using D ₃ 3-hexuccinate as a hapten has been disclosed. JP-A-53-87344 describes a radioimmunoassay method using 1α,25-dihydroxyvitamin D ₃ 25-hemisuccinate as a hapten.

Regarding these radioimmunoassay methods,
The antigen for the assay is one in which a hapten and a carrier protein are bonded via the carboxyl group at the 25th position or the carboxyl group in the substituent at the 3rd position of the hapten. In the case of such antigens,
The binding site between a hapten and a carrier protein is at position 25 of the hapten or near position 1.

In general, it is known that antigen antibodies (anti-hapten antibodies) for assay well recognize chemical structures on haptens that are distant from the carrier protein binding site.

Therefore, the anti-hapten antibody obtained using the hapten described in the above-mentioned reference is
The ability to recognize the structure at position 24 or position 24 may be impaired.

For such radioimmunoassay method, 25
It is not always possible to satisfactorily quantify active vitamin _D3 compounds having a hydroxyl group at the 1α, 1α or 24th positions.

Therefore, the present inventor conducted extensive research and developed a polymer compound obtained by using a novel vitamin D ₃ derivative having a carboxyl group or its ester group at the 22nd position as a hapten, and covalently bonding the hapten and an immunogenic carrier. As an antigen for producing antibodies for immunochemical measurement, antibodies obtained from this antigen are
We have discovered that the present invention is extremely useful as an antibody for enzyme immunoassays or radioimmunoassays, which are preferably applied to quantifying active vitamin _D3 compounds having a hydroxyl group at the 1α, 24th, or 25th positions. This is what I did.

Therefore, the present invention is based on the following formula [] [Here, R ₁ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms having a carboxyl group or an amino group, and R ₂ , R ₃ and R ₄ each independently represent a hydrogen atom or a hydroxyl group. ] This is an antigen for producing an antibody for immunochemical measurement, which is obtained by covalently bonding a vitamin D ₃ derivative represented by the formula and an immunogenic carrier via the carboxyl group or amino group of the vitamin D ₃ derivative.

In the present invention, the compound used as an antigen for producing an antibody for immunochemical measurement and a hapten for the antibody is a compound represented by the above formula [],
It has a characteristic structure of having a carboxyl group or its ertel group at the 22nd position.

In the above formula [], R ₁ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms and having a carboxyl group or an amino group. Here, examples of the alkyl group having 1 to 6 carbon atoms having a carboxyl group or an amino group include a carboxymethyl group, 2
- an alkyl group having 1 to 6 carbon atoms having a carboxyl group such as a carboxyether group, 3-carboxypropyl group, 4-carboxybutyl group, 5-carboxyheptyl group, 6-carboxyhexyl group;
Examples include alkyl groups having 1 to 6 carbon atoms and having an amino group such as aminomethyl group, 2-aminoethyl group, 3-aminopropyl group, 4-aminobutyl group, 5-aminoheptyl group, and 6-aminohexyl group. It will be done.

Among these, R ₁ is particularly preferably a hydrogen atom.

In the above formula [], R ₂ , R ₃ and R ₄ each independently represent a hydrogen atom or a hydroxyl group. Among others
Those in which R ₂ R ₃ are both hydroxyl groups are especially 24, 25
- Preferred as a hapten for assaying active vitamin _D3 such as dihydroxyvitamin _D3 .

Specific examples of the vitamin D ₃ derivatives represented by the above formula [] are as follows.

(1) 22-carboxy-24,25-dihydroxycholecalciferol, (2) 22-carboxy-1α,24,25-trihydroxycholecalciferol, (3) 22-carboxy-25-hydroxycholecalciferol role, (4) 22-carboxy-1α,25-dihydroxycholecalciferol, (5) 22-carboxy-1α,24-dihydroxycholecalciferol, (6) 22-carboxy-1α-hydroxycholecalciferol , (7) 22-(2-carboxyethoxycarbonyl)-
24,25-dihydroxycholecalciferol, (8) 22-(2-carboxyethoxycarbonyl)-
1α,24,25-trihydroxycholecalciferol, (9) 22-(2-carboxyethoxycarbonyl)-
1α,25-dihydroxycholecalciferol, (10) 22-(2-carboxyethoxycarbonyl)-
25-hydroxycholecalciferol, (11) 22-(2-carboxyethoxycarbonyl)-
1α-hydroxycholecalciferol, (12) 22-(2-aminoethoxycarbonyl)-24,
25-dihydroxycholecalciferol, (13) 22-(2-aminoethoxycarbonyl)-
1α,25-dihydroxycholecalciferol, (14) 22-(2-aminoethoxycarbonyl)-1α
-Hydroxycholecalciferol The above-mentioned vitamin D ₃ derivative of the present invention is produced as follows. In other words, the following formula [] [Here, R ₁ ′, R ₂ ′, R ₃ ′, R ₄ ′, and R ₅ are the same as the above definitions. ] It is produced by irradiating a cholesta-5,7-diene derivative represented by the following in an inert organic medium with ultraviolet rays, thermally isomerizing it, and then deprotecting it as necessary.

The starting compound cholesta-5,7-diene derivative represented by the above formula [] is the corresponding cholesta-5,7-diene derivative.
From the 5-ene derivative, this is brominated at the allylic position,
Produced by dehydrobromination.

In the above formula [], R ₁ ' is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms having a carboxyl group or an amino group, or a protected carboxyl group or a protected amino group. represents an alkyl group having 1 to 6 carbon atoms.

Here, examples of the alkyl group having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, butyl group, t-butyl group, heptyl group, and hexyl group. Examples of the alkyl group having 1 to 6 carbon atoms and having a carboxyl group or an amino group include those exemplified as R ₁ in the above formula []. 1 to 6 carbon atoms with a protected carboxyl group or a protected amino group
Examples of the alkyl group include the following. That is, in the alkyl group having 1 to 6 carbon atoms having a carboxyl group exemplified as R ₁ in the above formula [], the carboxyl group forms an ester bond, such as methoxycarbonylmethyl group, ethoxycarbonylmethyl group, butoxycarbonylmethyl group, etc. These include carbonylmethyl group, 2-methoxycarbonylethyl group, 2-ethoxycarbonylethyl group, and 2-butoxycarbonylethyl group. As the alkyl group having 1 to 6 carbon atoms having a protected amino group, in the alkyl group having 1 to 6 carbon atoms having an amino group exemplified as R ₁ in the above formula [],
The amino group is the group that formed the amide bond,
Examples include acetylaminomethyl group, 2-acetylaminoethyl group, and 3-acetylaminobutyl group.

In the above formula [], R ₂ ′, R ₃ ′, and R ₄ ′ each independently represent a hydrogen atom, a hydroxyl group, or a protected hydroxyl group. R ₅ represents a hydrogen atom or a protective group.
When R ₂ ′, R ₃ ′, and R ₄ ′ represent a protected hydroxyl group or R ₅ represents a protecting group, examples of the protecting group include acetyl group, propanoyl group, butanoyl group, pivaloyl group, pentanoyl group, and cyclohexanoyl group. aliphatic or aromatic carboxylic acid residues having 1 to 12 carbon atoms, such as chloroacetyl group, promoacetyl group, benzoyl group, p-promobenzoyl group, p-nitrobenzoyl group, ethylbenzoyl group, toluyl group, or the like; nitro, halogen, alkoxy substituted derivatives; or trimethylsilyl group,
Trialkylsilyl group such as dimethyl-t-butylsilyl group; or 2-tetrahydropyranyl group, 2
-2-cyclic ether groups such as -tetrahydrofuranyl group, etc. can be mentioned. Among these protective groups, acetyl group, propanoyl group, butanoyl group, bivanoyl group, pentanoyl group, cyclohexanoyl group, benzoyl group and the like are particularly preferred.

The cholesta-5,7-diene derivative represented by the above formula [] is thermally isomerized by irradiation with ultraviolet rays in an inert organic medium. The ultraviolet light used for ultraviolet irradiation is approximately 200 to 360 nm,
Preferably, those having a wavelength of 260 to 310 nm are used.

This ultraviolet irradiation reaction is carried out in an inert organic medium. Such inert organic media include, for example, hydrocarbons or halogenated hydrocarbons such as hexane, heptane, benzene, toluene, xylene, chlorobenzene, carbon tetrachloride; ethers such as diethyl ether, tetrahydrofuran, dioxane; or methanol, ethanol. Alcohols such as , propanol are preferably used.

Although the reaction temperature does not have a very important meaning in the ultraviolet irradiation reaction, it is usually carried out at -20°C to 120°C, especially -10°C to 50°C.

In this way, the carbon-carbon bonds at the 9 and 10 positions of the cholesta-5,7-diene derivative represented by the above formula [] are cleaved and converted into the corresponding previtamin D ₃ derivative. This previtamin D ₃ derivative is then isomerized by thermal energy to form a vitamin
becomes a _D3 derivative. This isomerization reaction is an equilibrium reaction between the previtamin D ₃ derivative and the vitamin D ₃ derivative, and the equilibrium value between the two differs depending on the reaction temperature.

In the present invention, the isomerization reaction is carried out at 20 to 120°C.
Preferably it is carried out at 40 to 100°C. Further, this isomerization reaction proceeds satisfactorily as it is in the inert organic medium used in the above-mentioned ultraviolet irradiation.

Therefore, for example, when the ultraviolet irradiation for producing the above-mentioned previtamin D ₃ derivative is carried out at 40°C, the previtamin D ₃ derivative produced at the same time as the cleavage of the carbon-carbon bonds at positions 9 and 10 progresses. In the reaction system, a reaction of isomerization to vitamin D ₃ derivatives will occur gradually. As is clear from the above, the isomerization reaction using thermal energy in the method of the present invention does not necessarily mean heating the reaction system.

Thus, the vitamin represented by the above formula []
A D ₃ derivative or a hydroxyl, carboxyl or amino protected derivative thereof is produced. hydroxyl group,
When a carboxyl group or an amino group is protected, it is necessary to remove the protecting group following the above-mentioned isomerization reaction. This deprotection reaction is a known reaction per se. For example, when the protecting group forms an acyl group, it is treated in an alkaline solution of a lower aliphatic alcohol such as methanol or ethanol, or LiAIH ₄ etc. in ether. It can be treated with metal hydride. Temperature: -10℃
~50℃ is sufficient.

When the protecting group is bonded to the oxygen atom of the hydroxyl group to form an ether group, it can be easily removed cyclically or by contacting with an acid or an alkali.

Thus, the vitamin represented by the above formula []
A D ₃ derivative is obtained.

In the vitamin D ₃ derivative represented by the above formula [], when R ₁ represents an alkyl group having 1 to 6 carbon atoms having a carboxyl group or an amino group, the vitamin D ₃ derivative can also be obtained by the following method. That is, a vitamin D ₃ derivative in which R ₁ is a hydrogen atom in the above formula [] and a carbon number 1 to 6 having a carboxyl group such as glycolic acid, lactic acid, β-hydroxypropionic acid, malic acid, etc.
It can also be produced by carrying out an esterification reaction with a hydroxy acid or an amine compound having 1 to 6 carbon atoms and having an amino group such as 2-hydroxyethylamine or 3-hydroxybutylamine.

The antigen for producing antibodies for immunochemical assay of the present invention is obtained by covalently bonding the vitamin D ₃ derivative of the above formula [] and an immunogenic carrier via the carboxyl group or amino group of the vitamin D ₃ derivative. It is what it is.

Immunogenic carriers used to obtain antigens by covalent bonding with vitamin D ₃ derivatives include proteins, polypeptides, glycoproteins, and the like. Examples of such proteins include bovine serum albumin (BSA), human serum albumin, human gamma globulin, methylated bovine serum albumin, rabbit serum albumin, bovine gamma globulin, and hemocyanin. Examples of the polypeptide include polylysine, poly-6-lysine-polyglutamic acid copolymer, and the like. Glycoproteins include lipopolysaccharide, ficoll,
Examples include keyhole hemocyanin. Among these, proteins are preferred, with bovine serum albumin and human serum albumin being particularly preferred.

Such an immunogenic carrier and the vitamin of the above formula []
A covalent bond with the D ₃ derivative is formed directly or indirectly with the functional group of the immunogenic carrier via the carboxyl group or amino group at position 22 of the vitamin D ₃ derivative. Examples of the functional group of the immunogenic carrier include an amino group of lysine, a carboxyl group or amino group of aspartic acid or glutamic acid, a phenolic hydroxyl group in a tyrosine residue, and a mercapto group of cysteine.

As a method for covalent bonding, a method of reacting N-hydroxysuccinimide, alkyl chloroformate, etc. with the carboxyl group or amino group of the vitamin D ₃ derivative to activate the carboxyl group or amino group of the vitamin D ₃ derivative and then bonding; A method using a condensing agent such as 1-ethyl-3-(3-dimethylaminopropyl)-carboximide hydrochloride, dicyclohexylcarbodiimide, N-nityl-5-phenylisoxazolium-3'-sulfonate; or a method using these Examples include a method of using both methods together. Among these, a method in which an activating agent such as N-hydroxysuccinimide and a condensing agent such as dicyclohexylcarbodiimide are used in combination is preferred.

To give an example of a preferred embodiment, for example, 22-carboxy-24,25-dihydroxycholecalciferol, N-hydroxysuccinimide, and dicyclohexylcarbodiimide are dissolved in tetrahydrofuran, and left at room temperature for more than ten hours to dissolve 22-carboxy There is a method in which N-hydroxysuccinimide ester of -24,25-dihydroxycholecalciferol is produced and then this is reacted with human serum albumin in a phosphate buffer.

Vitamin D ₃ conjugated to an immunogenic carrier
The number of derivative molecules is 5 to 5 per molecule of immunogenic carrier.
20 pieces is preferred. In particular, 5 to 10 pieces are preferable.

The antigen for producing antibodies for immunochemical measurements thus obtained is inoculated into a host animal to induce antibodies, and used for antibody production.

Examples of host animals include domestic rabbits, rats, cows,
Examples include warm-blooded animals such as sheep. Parenteral administration is used to inoculate such animals;
For example, it is carried out by subcutaneous or intradermal injection.

Freund's complete adjuvant is usually used for inoculation.

The method of inoculation is 200 μg to 500 μg of antigen.
This is injected subcutaneously or intradermally, and the same antigen is re-injected every 4 weeks, during which time the blood antibody titer is measured every 10 days and whole blood is collected at the highest point. Serum can also be obtained from collected blood and used as it is as an antibody. Preferably, by means such as ammonium sulfate fractionation or gel filtration method.
Antibodies can be prepared by obtaining an IgG fraction.

Each of the antibodies obtained in this way has the 1α position,
Active vitamin D ₃ having a hydroxyl group at the 24th or 25th position, such as 1α-hydroxyvitamin D ₃ , 25-
Can recognize compounds such as hydroxyvitamin D ₃ , 1α,25-hydroxyvitamin D ₃ , 24,25-dihydroxyvitamin D ₃ and is therefore useful as a pore for enzyme or radioimmunoassays. It is.

When such an antibody is used for enzyme immunoassay, it is used as a reagent in measurement kit b together with an enzyme-labeled antigen.

The enzyme-labeled antigen mentioned here includes the vitamin D ₃ derivative represented by the above formula [] and enzymes such as BD-galactosidase, alkaline phosphatase, glucose oxidase, lipase,
Enzymes such as peroxidase, preferably B-D
- Examples include those obtained by covalently bonding enzymes such as galactosidase and alkaline phosphatase.

When using the antibody of the present invention for radioimmunoassay, ³ H (tritium), ¹⁴ C (carbon 14)
It is used as the reagent b of the measurement kit together with 24,25-dihydroxyvitamin D ₃ , 25-hydroxyvitamin D ₃ , 1α-hydroxyvitamin D ₃ , and 1α,25-dihydroxyvitamin D ₃ radiolabeled with eg. Alternatively, the antibody can be used together with ¹²⁵ I-Protein A radiolabeled ^with 125 I as a reagent for radioimmunoassay called the Sand-Deutsch method.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Reference example 1 (i) Synthesis of 22-methoxycarbonyl-3β,24-diacetoxycholest-5,7-dien-25-ol 22-methoxycarbonyl-3β,24-diacetoxycholest-5,7-ene −25−all
450 mg was dissolved in 10 ml of carbon tetrachloride. To the resulting solution was added 137.7 mg of 1,3-dibromo-5,5-dimethylhydantoin under a nitrogen atmosphere at a bath temperature of 95° C. while heating and stirring. After the addition, the mixture was vigorously heated under reflux and stirred while being irradiated with an infrared lamp, and the bromination reaction was carried out for 15 minutes. After the reaction is complete,
The reaction solution was filtered and then concentrated to obtain a concentrated residue containing the 7-bromine compound of 22-methoxycarbonyl-3β,24-acetoxycholest-5-en-25-ol.

The obtained residue was dissolved in 12 ml of xylene, and added dropwise to a mixed solution of 7.5 ml of xylene and 2.5 ml of s-collidine while heating and stirring at a bath temperature of 170°C. After dripping,
The reaction was completed by heating and stirring for an additional 20 minutes.

After the reaction was completed, the mixture was diluted with an appropriate amount of water and extracted with ethyl acetate. Add ethyl acetate extract to 1N hydrochloric acid,
After washing with an aqueous sodium bicarbonate solution and saturated saline, drying over anhydrous sodium sulfate, filtering, and concentration, 22-methoxycarbonyl-3β,
24-diacetoxycholester-5,7-diene-
A concentrated residue containing 25-ol was obtained.

The obtained concentrated residue is subjected to silica gel thin layer chromatography (solvent benzene-ethyl acetate system)
Possibly 22-methoxycarbonyl-3β,24
-diacetoxycholester-5,7-diene-25
-ol 154 mg (yield 35%) was obtained. The physical properties of this product were as follows.

UV (λ ^EtoH _{nax on} ): 293, 281, 271, 262 (shoulder) High resolution mass spectrum: M ⁺ −AcOH, 498.3358 (calculated value C ₃₁ H ₄₆ O ₅ = 498.3345) (ii) 22-methoxycarbonyl-3β ,24-diacetoxy-25-hydroxyvitamin D ₃ 22-methoxycarbonyl-3β,24-diacetoxycholester-5,7-dien-25-ol
100 mg was dissolved in 600 ml of deoxygenated benzene. The resulting solution was irradiated using a 200 watt Hanobia lamp surrounded by a Vycor filter for 7.5 minutes under controlled stirring at 5°C. This cold solution was concentrated under reduced pressure to about 100 ml while controlling the temperature at 30°C. The resulting benzene solution was heated under reflux for 2.5 hours to complete the reaction. After the reaction was completed, the reaction solution was concentrated under reduced pressure while controlling the temperature at 30°C. The obtained residue is 2
22-methoxycarbonyl- 25.5 mg of 3β,24-diacetoxy-25-hydroxyvitamin D ₃ was obtained. The physical properties of this product were as follows.

UV (EtOH; nm): λmax, 263 (ε=15500) λmin, 228 (ε=8200) MS (m/e): 558 (M ⁺ ), 198, 281, 253, 158, 118 High resolution mass spectrum: M ⁺ 558, 3660 (calculated value C ₃₃ H ₅₀ O ₇ = 558.3559) (iii) Synthesis of 22-carboxyl-24,25-dihydroxyvitamin D ₃ 22-methoxycarbonyl-3β,24-diacetoxy-25-hydroxyvitamin D ₃ Dissolve 14 mg in 2 ml of tetrahydrofuran, add 2 ml of 10% potassium hydroxide-methanol, and dissolve under nitrogen atmosphere.
The mixture was heated and stirred at 65°C for 2 and a half hours. 0.25 ml of acetic acid was added and extracted with ethyl acetate. After washing with saturated brine, it was dried over anhydrous sodium sulfate. 22-carboxyl-
9.8 mg of 24,25-dihydroxyvitamin _D3 was obtained.
The physical properties of this product were as follows.

UV (EtOH; nm): λmax 263nm (ε=15700) λmin 227.5nm (ε=8300) MS (m/e): 442 (M ⁺ −H ₂ O), 424, 406, 136, 118 High resolution mass spectrum : M ⁺ −H ₂ O 442.3117 (calculated value C ₂₈ H ₄₂ O ₂ = 442.3083) Example 1 Production of 22-carboxy-24,25-dihydroxycholecalciferol-HSA conjugate: (i) 22-carboxy- Production of N-hydroxysuccinimide ester of 24,25-dihydroxycholecalciferol 22-carboxy-24,25-dihydroxycholecalciferol (1.0×10 ^-6 mol), N-hydroxysuccinimide (1.5×10 ^-6 mol) ) and dicyclohexylcarbodiimide (DCC, 1.5×
10 ^-6 mol) was dissolved in tetrahydrofuran (THF, 900μ) and stirred in the dark for 12 hours. The resulting dicyclohexylurea precipitate was centrifuged (10,000 g x 10 min) and then concentrated under reduced pressure. The resulting N-hydroxysuccinimide ester was dissolved in THF (200μ).

(ii) N-hydroxysuccinimide ester and
Reaction with HSA.

N-hydroxysuccinimide ester dissolved in THF (200μ), HSA (50μ) dissolved in 0.05M phosphate buffer (PH7.4, 500μ)
mg) and pyridine (100μ) at 4°C.
The mixture was stirred for 12 hours. THF (200μ) was then added to the resulting reaction mixture and the cooled 50%
Dialysis was performed against THF/water for 12 hours and then against cold water for 48 hours.

Thus, a 22-carboxy-24,25-dihydroxycholecalciferol-HSA conjugate was obtained.

Reference Example 2 Rabbits were immunized with 22-carboxy-24,25-dihydroxycholecalciferol-HSA conjugate as an antigen and antibodies were obtained as follows.

22-carboxy-24,25-dihydroxycholecalciferol-HSA conjugate (200μg)
was dissolved in physiological saline (100 μg) and added to Freund's Complete Adjuvant (Freund's
Complete Adjuvant) was administered intradermally to male rabbits for initial immunization. Subsequent immunization was carried out 4, 8 and 12 weeks later, and antiserum usable for radioimmunoassay or enzyme immunoassay was obtained approximately 4 months after the start of immunization.

Reference Example 3 (i) Using the following reagents, it was investigated whether the antibody obtained in Reference Example 2 could recognize vitamin D ₃ by the method described below.

〔reagent〕

¹²⁵ I-labeled protein A (8.32 nCi/ng
protein), antigen: scumylation-vitamin _D3 -BSA,
Microliter plate, RIA buffer A buffer: 0.05M phosphate buffer PH7.4, 0.1%
_NaN3 , B buffer: 0.05 phosphate buffer PH7.4, 1.0% BSA,
0.1% _NaN3 , C buffer: 0.05M phosphate buffer PH7.4, 0.1% Tween 20, 0.1% _NaN3 [Method] (A) Antigen (succinylated vitamin _D3 -BSA)
was dissolved in buffer A to give a concentration of approximately 20 μg/ml protein.

Antigen solutions (Antigen aliqvot, 100μ) of various concentrations obtained using this stock solution and buffer A were placed in wells and incubated at 37° C. for 1 hour.

(B) The antigen solution was removed and B buffer (0.2 ml) was added to each well and incubated at 37°C for 1 hour. Then, the B buffer was removed, washed with C buffer, and dried.

(C) Add 100μ of antibody to each well and incubate at 4°C for 12
time incubation. The solution was then removed, washed with C buffer and dried.

(D) ¹²⁵ I-labeled protein A solution (250n
Solutions of various concentrations (100μ) prepared from 10g/ml) were added to each well. This was followed by incubation for 22-24 hours at room temperature.

(E) After removing the solution from each well, washing with C buffer and drying, radioactivity was measured using an r-counter.

Since a predetermined count number was obtained by r-counter measurement, ¹²⁵ I-Protein A was bound to the vitamin D ₃ -antibody conjugate, and therefore 22-carboxy-24,25- obtained in Example 2 Dihydroxycholecalciferol – Antibodies obtained from HSA are vitamins
It was confirmed that _D3 can be recognized.

(ii) Using the reagent used in (i), as the test compound
A calibration curve for radioimmunoassay using ¹²⁵ I-protein A was prepared using 1α,25-dihydroxyvitamin D ₃ in the following manner.

〔Method〕

(A) Antigen (succinylated vitamin D ₃ -BSA)
was dissolved in buffer A, and antigen solutions (100μ) of various concentrations from this solution and buffer A were added to wells and incubated at 37°C for 1 hour.

(B) Remove the antigen solution, add B buffer (0.2 ml), and incubate at 37°C for 1 hour.
The solution was then removed, washed with C buffer and A buffer, and then dried.

(C) Antibody (100 μ) dissolved in buffer A and 1α,
25-dihydroxyvitamin D ₃ solution (10μ)
was added to each well and incubated at 4°C for 12 hours. The solution was then removed, washed with C buffer and B buffer, and dried.

(D) ¹²⁵ I-labeled protein A solution (250n
Solutions (100μ) of various concentrations adjusted from
time incubation.

(E) The solution was removed from each well, washed with C buffer, dried, and then radioactivity was measured with an r-counter.

By the above method, the concentration of 1α,25-dihydroxyvitamin D ₃ was varied, and the ratio of 1α,25-dihydroxyvitamin D ₃ to the number of counts on the r-counter when 1α,25-dihydroxyvitamin D ₃ was not added was determined. A calibration curve was created by determining the ratio of counts when ¹²⁵ - specific radioactivity (B/Bo%) was added. According to this calibration curve, the dilution rate is 9600
By using 1x antibody and ^10-13x dilution of antigen, 1α, in the range 10f-1p (mol/well),
It has been demonstrated that 25-dihydroxy-vitamin D ₃ can be quantified favorably.

Claims (1)

[Claims] 1. The following formula [] [Here, R ₁ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms having a carboxyl group or an amino group, and R ₂ , R ₃ and R ₄ each independently represent a hydrogen atom or a hydroxyl group. ] An antigen for producing an antibody for immunochemical measurement, which is obtained by covalently bonding a vitamin D ₃ derivative represented by the following formula to an immunogenic carrier via a carboxyl group or an amino group of the vitamin D ₃ derivative. 2. The antigen according to claim 1, wherein in the above formula [ ], R ₁ is a hydrogen atom. 3. The antigen according to claim 1 or 2, wherein in the above formula [], both R ₂ and R ₃ are hydroxyl groups. 4. The antigen according to any one of claims 1 to 3, wherein the immunogenic carrier is a protein. 5 The immunogenic carrier is bovine serum albumin (BSA)
or human serum albumin (HSA).