JPH02104570A - Novel 1alpha,25-dihydroxyvitamin d3 derivative and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound shown by formula I [one of R1 and R2 is OH and the other is -O-CO-R3-NH2 (R3 is 1-10C alkylene) and the title radioisotope compound shown by formula II [one of R1 and R2 is OH and the other is -O-CO- R3-R5 (R3 is 1-10C alkylene; R5 is residue containing radioisotope of iodine)]. USE:Determination of 1alpha,25-dihydroxyvitamin D3, an active type in a specimen solution. An antibody prepared by bonding a compound sown by formula I to a carrier protein as a hapten and immunizing and a compound shown by formula II are used. PREPARATION:1alpha,25-dihydroxyvitamin D3 is reacted with an amino acid containing protected amino group, the amino-protecting group is eliminated from the prepared compound to give a compound shown by formula I. Radioactive iodine (preferably <125>I) is labeled with the compound shown by formula I by indirect labeling to give a compound shown by formula II.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention provides novel lα,25-dihydroxyvitamin D
, an antibody obtained by synthesizing an amino acid derivative, conjugating it as a hapten with a carrier protein, and immunizing it, and a novel iodine radioisotope-labeled 1α,25-dihydroxyvitamin D3.
Using derivatives, the active form of 1α, 25-
This invention was established for the purpose of quantifying the amount of dihydroxyvitamin D3, and relates to the fields of synthetic chemistry, immunochemistry, and radiochemistry.

[Conventional technology]

Conventionally, radioreceptor assay (RRA) and radioimmunoassay (RIA) have been reported as methods for quantifying 1α,25-dihydroxyvitamin D3, and the labeled compound in these assays is tritium [
3H] labeled compound is used. The receptors used in the RRA method are generally those derived from the cytoplasm of the chicken small intestine (Arch,
Biochem, Biophys, 176.235~
243 (1976); Vitamin Vol. 55 No. 12 (12
) 595-605 (1981), Clinical Examination 26,
1 (January) 7-18 (1982)). Furthermore, as haptens for producing antibodies used in the RIA method, those having a carbonyl group at the end of the side chain are known (C1inicalScience and
Molecular Medicine, 5↓, 329~
332 (1978);Clncal Chem,
, 27/3.458-463 (1981); 1bi
d, 2615.562-567 (1980); 1
bidi29/1, 196-200 (1983) iJ
.

Biochem, 98,991-998 (1985
);Biochem,Biophys,Res,,Co
mm, 431-436 (1983)
476'53; JP 58-193463; JP 58-92656; JP 59-148
No. 775; JP-A-61-48497].

In any quantitative method, the labeled compound is 1α
, 25-dihydroxyvitamin D3 tritium [3H
] They have in common that they use a labeled compound.

[Problem to be solved by the invention]

In the determination of 1α,25-dihydroxyvitamin D in the test solution, the previously used tritium ('
H) The labeled compound has radiation energy of 32p or 1
25 It is extremely low compared to 1, and therefore quantitative methods using tritium [3H] labeled compounds require a detection device such as a liquid scintillation counter.
It was expensive and the measurement operation was complicated. Therefore, a highly sensitive and simpler measurement method has been desired.

[Means to solve the problem]

In view of the above-mentioned problems, the present inventors conducted intensive research and developed a radioactive iodine-labeled 1α,25-dihydroxyvitamin D3 derivative that has high radiation energy and is excellent in versatility. Tritium [3H] is a β-ray emitting nuclide with conventionally low radiation energy per unit time.
There were various vitamin D3 labeled compounds. However, like tritium [3H], vitamin D3 derivatives labeled with iodine (1251), a gamma ray-emitting nuclide with high radiation energy commonly used in radioimmunoassays, have a conjugated triene structure that is unstable, which is a characteristic of vitamin D. Therefore, it is thought to induce radical reactions and overcome self-decomposition.
Not reported to date. However, the iodine [12'-labeled 1α,25-
It has been found that dihydroxyvitamin D, a derivative, is stable and can be used in radioimmunoassays without causing structural decomposition by gamma rays.

In general, radioactive iodine labeling methods for low molecular weight compounds include direct labeling method (chloramine-T method) and indirect labeling method (method using Porton-Hunter reagent) (Amersham
; Amersham Note 1N0V, 1981), L Kashi,
Since vitamin D is highly unstable to acids, oxygen, oxidizing agents, heat, and light, it is most preferable to use indirect labeling methods that proceed under mild reaction conditions. In carrying out the indirect labeling method, first, a novel compound having an amino group at the end of the side chain was used.
An α,25-dihydroxyvitamin D3 amino acid derivative was synthesized. Furthermore, this derivative was combined with a carrier protein as a hubten, and this was inoculated into animals to obtain antibodies. The derivative was also labeled with iodine radioisotope. Furthermore, we established a radioimmunoassay measurement system using the obtained antibody and an iodine radioisotope-labeled compound, and found that 1α,25-dihydroxyvitamin D in the test solution was
3 (hereinafter sometimes abbreviated as 1α, 25 (OH)z D:l), we found that the assay method using this measurement system is a highly sensitive, simple, and extremely useful quantitative method. .

It has also been found that 1α,25-dihydroxyvitamin D3 can be quantitatively measured with high sensitivity using the iodine radioisotope-labeled compound of the present invention by the Competitive Protein Affair Method (CPBA method).

That is, the present invention was completed based on the above findings, and is based on the formula (1) [wherein, one of R+ and Rz is a hydroxyl group and the other is -O
Co R2NH2, R1 represents an alkylene group having 1 to 10 carbon atoms] and a novel 1α,25-dihydroxyvitamin D3 amino acid derivative represented by the formula One is a hydroxyl group and the other is -0-CO-R3N H-R4, and R1 has 1 carbon number.
~10 alkylene groups, R4 represents an amino-protecting group] is characterized by removing the amino-protecting group from a 1α,25-dihydroxyvitamin D derivative represented by: A new 1α represented by 2 (I)
, 25-dihydroxyvitamin D, an amino acid derivative of the formula (III) [wherein, one of R and R2 is a hydroxyl group and the other is -0
-Co-Rs-NH-R5, R1 is the number of carbon atoms ~ 1
This is an iodine radioisotope-labeled 1α,25-dihydroxyvitamin D3 antibody conductor represented by the following formula. In addition, the test solution contains iodine radioisotope labeled tillα,25-dihydroxyvitamin D represented by formula (III), a derivative and lα,25-dihydroxyvitamin D3.
A radioisotope-labeled 1α,25-dihydroxyvitamin D derivative of the iodine produced by reacting with an antibody (hereinafter sometimes abbreviated as antibody) or vitamin D binding protein (hereinafter sometimes abbreviated as DBP) □Antibody conjugate or radioisotope labeling of the iodine lα,25-dihydroxyvitamin D3 derivative □DBP
conjugate and radioisotope labeling of the unreacted iodine 1α.

25-dihydroxyvitamin D3 derivative and then quantifying the amount of labeled iodine, which is one of the separated radioactive isotopes.
This is a method for quantifying α,25-dihydroxyvitamin D3.

The present invention will be explained in detail below.

First, the 1α,25-dihydroxyvitamin D3 amino acid derivative represented by formula (I) is a 1α,25-dihydroxyvitamin D3 amino acid derivative.
α hydroxyl group or 3 of carbon 1 of dihydroxyvitamin D3
It is obtained by covalently bonding through the β-hydroxyl group at the carbon position, and RI in formula (1) is an alkylene group having 1 to IO carbon atoms. In addition, the 1α,25-dihydroxyvitamin D and amino acid derivative represented by this formula (1) are the 1α
.

It is also used as the Fm form of habten when producing a 25-dihydroxyvitamin D3 antibody, and the chain length of the crosslinking group when bonding to a carrier protein as a hapten derivative is preferably 1 to 10 carbon atoms, particularly preferably Since 2 to 6 are mentioned, it is particularly preferable that R1 is a linear alkylene group having 2 to 6 carbon atoms.

1α,25-dihydroxyvitamin D3 represented by formula (T)
Amino acid derivatives are produced by reacting 1α,25-dihydroxyvitamin D3 with an amino acid with a protected amino group, resulting in 1α,25-dihydroxyvitamin D represented by formula (U).
3 dioptricate is obtained, and the derivative ([1) is obtained by removing the amino protecting group.

First, the raw amino acid used to protect the amino group is one represented by the following formula (rV) HOOC-Ri-NH2(IV) (wherein R3 represents the same group as above), The alkylene group represented by R3 has n-1+ carbon atoms, glycine, n=2. β-alanine, n-3, γ-amino-n-butyric acid, n=4. δ-aminopentaic acid, n=5. ε−
amino-n-caproic acid, n=6.7-aminohebbutanoic acid, and further n=10.11-aminoundecylic acid, wherein the carbon structure in the amino acid is preferably linear;
Those having an alkylene group having 2 to 6 carbon atoms are preferred, and from the perspective of the present invention, δ-amino-levulinic acid,
Glycylglycine, D- and/or L-form lysine which is a δ-amino acid, etc. can also be used. Furthermore, amino acids having a hydroxyl group in the side chain, such as 4-amino-3-hydroxybutyric acid, can also be used in the same way by protecting the hydroxyl group.

In addition, as an amino protecting group for amino acids, amino protecting group
! When removing groups, if severe removal conditions are required, vitamin DH itself may be decomposed. For example, 9-fluorenylmethyloxycarbonyl group (hereinafter sometimes abbreviated as Fmoc), 9-
Examples include (2-sulfo)fluorenylmethyloxycarbonyl group, 1,■-dimethyl-2-cyanoethyloxycarbonyl group, 5-benzisoxazolylmethyloxycarbonyl group, etc., but particularly preferred are amino acid A 9-fluorenylmethyloxycarbonyl group capable of protecting a group is good as a protecting group for an amino group. To obtain amino acids with protected amino groups,
Examples of active derivatives having an amino protecting group as mentioned above include Fm.

Examples include N-(9-fluorenylmethyloxycarbonyloxy)succinimide, which is an active ester having C, and 9-fluorenylmethylchloroformate can also be used. In addition, when protecting an amino group, the active derivative and the desired amino acid are combined using a known method (Carpin et al.

and G, Y., Han J., Org., Chem.
, 37. 3404 (1972)) to obtain Fmoc-amino acids.

Furthermore, Fmoc-
Taking as an example a method for obtaining the lα,25-dihydroxyvitamin D1 amino acid derivative of formula (II) using an amino acid, F m o c-amino acid is used as the α of the 1st carbon of lα,25-dihydroxyvitamin D. Upon covalent bonding via a hydroxyl group or a β-hydroxyl group at the 3-position carbon, 3α,
F m for 1 equivalent of 25-dihydroxyvitamin D
A compound represented by formula (II) can be obtained by using one equivalent of a commonly used carboxyl group-reactive derivative such as oc-amino acid or its acid anhydride, acid halide or active ester. More preferably, a mixed acid anhydride consisting of 1 equivalent of Fmoc-amino acid and 1 equivalent of "other acid" is reacted in an anhydrous solvent in the presence of a base under an inert gas atmosphere. The mixed acid anhydride is used to react with 1α,25-dihydroxyvitamin D3 by adding Fmo to the hydroxyl group at carbon 25 position of 1α,25-dihydroxyvitamin D3.
This is to prevent the formation of by-products in which c-amino acids are bonded. Valeric acid, pivalic acid, and isobutyloxycarboxylic acid are used as "other acids" when preparing the mixed acid anhydride, and in particular, pivalic acid is used. Acids are preferred. 1α, 25
- In the reaction between dihydroxyvitamin D3 and Fmoc-amino acid, the anhydrous solvent is tetrahydrofuran,
Anhydrous organic solvents such as dioxane are preferred. Furthermore, as the base, dimethylaminopyridine (DMAP) and piperidinoviridine (PPY) are particularly preferred. this is,
Useful for esterification of sterically hindered secondary and tertiary alcohols. Argon and nitrogen gas are preferred as the inert gas. Further, during the reaction, the reaction temperature is preferably 0 to 20°C and the reaction time is preferably 1 to 3 hours. lα, 25 expressed by the formula (I[) obtained in this way
The -dihydroxyvitamin D3 derivative has the following formula [V] -0-QC-R, -NH-R, (V) (wherein R3 and R4 are It is a mixture of compounds in which residues represented by (the same groups as above) are covalently bonded, and it can be separated and purified by HPLC or the like if necessary.

Furthermore, the lα,25-dihydroxyvitamin D3 derivative represented by formula (II) was converted into a compound of formula [
1α,25-dihydroxyvitamin D represented by 1),
Use it as an amino acid derivative.

In performing this protective group removal operation, the base used includes piperidine, morpholine, ethanolamine, etc., with morpholine being particularly preferred, and the inert solvent preferably being ethanol and methanol, particularly anhydrous ethanol and anhydrous methanol. is preferred. The reaction was carried out under an inert gas atmosphere, shielded from light, and the reaction temperature was 0 to 20°C.
The reaction time is preferably 1 to 3 hours. As the 1α,25-dihydroxyvitamin D3 derivative represented by formula (II), a mixture of compounds in which a residue represented by formula (V) is covalently bonded to the 1α or 3β position of 1α,25-dihydroxyvitamin D3 is used. When used, the correspondingly produced mixture of two species represented by formula (T) may be isolated and purified by HPLC or the like, if necessary. Furthermore, when a compound represented by the formula [■] isolated and purified in the previous step is used, it may be purified using a purification means such as column chromatography or TLC, if necessary.

In addition, 1α is produced as a by-product in the above-mentioned synthesis process.

A compound in which residues represented by formula [v] are covalently bonded to the 1α and 3β positions of 25-dihydroxyvitamin D can be obtained by first hydrolyzing the raw material in an alkaline aqueous solution.
, 25-dihydroxyvitamin D. Furthermore, by treating the above-mentioned by-product compound with morpholine, the formula (Vr) [wherein R6 and R7 are -0CR1-
NH2 and R3 is the same group as above] is obtained, and can also be used as a hapten compound.

Next, the iodine radioisotope-labeled lα,25-dihydroxyvitamin D3 derivative represented by formula [l11] will be described. R3 has the same meaning as R1 explained in the above formula (1), R3 is a residue having a radioactive isotope of iodine, and the radioactive isotope of iodine used is +251, 1311, etc. However, 125I, which has a relatively long half-life, is preferred.

As a residue having a radioactive isotope of iodine [12SI], 3-(4-hydroxy-3-iodo(+251)
phenyl)propionyl M, 3-(3,5-short(12'I)-4-hydroxyphenyl)propionyl group, 2-(4-hydroxy-3-iodo [125r)
phenyl)acetyl group, 2-(3,5-short(”
J)-4-hydroxyphenyl)7-t=thi/L4.2
-Iodo (+25 triacetyl group, 4-iodo (12%
l)-benzoxymethylcarbonyl group, N-substituted-
Examples include 3-iodo (1251) tyrosine residue. In particular, 3-(4-hydroxy-3-iodo (Its t
) phenyl) propionyl group and 3- (3,5-
A short [Izs □-4-hydroxyphenyl)propionyl group is preferred.

In the production of radioisotope-labeled 1α,25-dihydroxyvitamin D derivatives of iodine, the 1α,25-dihydroxyvitamin D3 amino acid derivative represented by formula (1) is labeled with an indirect labeling method (for example, using the Porton-Hunter reagent). By labeling with radioactive iodine according to method), iodine is labeled with a radioactive isotope 1α represented by the formula [■].

A 25-dihydroxyvitamin D3 derivative is obtained.

The indirect labeling method referred to here refers to the reactive derivative described below [■] whose constituent component is a residue having a radioactive isotope of iodine.
The formula (I
II) Iodine radioisotope labeling 1α, 2
5-dihydroxyvitamin D3B The above-mentioned reactive derivative [■] is represented by the formula % ( % formula % ( ), R2 has the same meaning as above, and
Oxy group, 5-norbornene-2.

It represents a 3-dicarboxygomideyl-N-oxy group or a maleimidyl-N-oxy group, R3 is a 3-(4-hydroxy-3-iodo(+2% I)phenyl)propionyl group, and X is a succinylimidyl group. -N-oxy group, N-succinylimidyl 3-(4-hydroxy-3-:l?-F(same phenyl)propionate) is commercially available as (last) Polton-Hunter reagent, and Can be used.

For example, in order to indirectly label 1α,25-dihydroxyvitamin D, amine 28m8 body represented by formula (1) using this [Izs 1) Porton-Hunter reagent, a radioactive isotope of iodine and an amino group are used. When covalently bonding via iodine (Its 1) used in the reaction, 1α. It is preferable to add the 25-dihydroxyvitamin D3 amino acid Ltd, jIL form [I] in an excess of about 500 to 2000 times, particularly preferably 1000 times. Usually, the reaction is carried out at 0-30℃, 12-72℃
All you have to do is take your time. Here, the reaction product iodine (1
The radioisotope-labeled 1α,25-dihydroxyvitamin D3g3 form (III) of 2S I) is purified by TLC or high-performance liquid chromatography (HPLC) to increase its utilization in the tA method or CPBA method. It is preferable to The thus obtained iodine (Its 1 )-labeled 1α,25-dihydroxyvitamin D, derivative (I[I] has a half-life of 2 months, which is the half-life of iodine (Its 1 ), when stored in an ethanol solution at -20°C. The product was stable and could be used for radioimmunoassays.In this case, the storage temperature should be as low as possible, preferably from 5°C to -20°C, especially -20°C.
℃ or less is preferable. The solvent is preferably alcohol-based or ether-based, and during storage, it is preferable to replace the solvent with inert gas.

Next, the 1α,25-dihydroxyvitamin D3 amino acid HA” expressed by the formula (1) obtained as described above, which is used for producing an antibody against the hapten 1α,25-dihydroxyvitamin D3, is used as a carrier. A 1α,25-dihydroxyvitamin D3 antibody can be produced by binding it to a protein and inoculating it into an animal.In order to obtain a 1α,25-dihydroxyvitamin D3 antibody, first the immunogenicity of Habten used for antibody production is determined. Examples of carrier proteins necessary to obtain antigens include simple proteins, polypeptides, complex proteins such as glycoproteins, and examples of simple proteins include bovine serum albumin (BSA), human serum albumin, and human serum globulin. Examples of polypeptides include polylysine, and examples of F''fH white include mucoprotein. Among these, simple proteins are preferred;
Particularly preferred are bovine serum albumin and human serum albumin. 1α,25-dihydroxyvitamin D, amino acid derivative (1) and the above-mentioned carrier protein are covalently bonded in the presence of a condensing agent and a crosslinking agent. In this case, dicyclohexylcarbodiimide (DCC), acid anhydride, glutaraldehyde, etc. are used as the condensing agent and crosslinking agent, and DCC is particularly preferred. The binding ratio of 1α,25-dihydroxyvitamin D, amino acid derivative and carrier protein is:
In general, if the amount is too high or too low, the titer of the antibody obtained will decrease, so use 1α per molecule of carrier protein.

10 25-dihydroxyvitamin D3 amino acid derivatives
It is preferable to combine up to 40 pieces.

The above-described conjugate for producing antibodies thus obtained can be inoculated into animals to produce antibodies. To inoculate animals, parenteral administration methods are employed, such as by subcutaneous or subcutaneous injection. When inoculating, for example, lα,2 obtained by the above procedure
The conjugated antigen of 5-dihydroxyvitamin D, amino acid derivative-carrier protein was dissolved in a buffer solution or physiological saline, and the same amount of complete Freund's adjuvant (Com
Plete freund's adjuvant:
After adding C, F, and A) and sufficiently emulsifying, the animal is subcutaneously administered to the infected animal to be used, and the same binding antigen is administered approximately 10 times every 1 to 3 weeks for immunization. Furthermore, if necessary, the bound antigen may be directly inoculated into the lung, which is the central organ responsible for immunity. During this period, blood antibody titers are measured at regular intervals, and whole blood is collected at the highest point, allowed to stand, coagulated, and centrifuged to obtain antiserum containing 1α,25-dihydroxyvitamin D and antibodies. Further, as the warm-blooded animal used in this case, any animal capable of producing antibodies may be used, and in order to obtain a large amount of antibodies, it is preferable to use large animals such as goats, cows, and sheep. Chickens, rabbits, rats, and mice are usually used, but there are no limitations. In addition, lα obtained from these immunized animals
, 25-dihydroxyvitamin D, 1α,25-dihydroxyvitamin D3 antibody can be obtained from antiserum containing antibodies by a commonly used antibody purification method, such as ammonium sulfate fractionation of the antiserum, Then, it may be purified and collected by ion exchange chromatography or gel filtration.

Furthermore, as a method for obtaining 1α,25-dihydroxyvitamin D3 antibodies, target antibody-producing lung cells of animals inoculated with the above-mentioned 1α,25-dihydroxyvitamin D3 amino acid derivative-carrier protein conjugate antigen and established bone marrow cells are used. A method for producing monoclonal antibodies produced by hybridomas (fused cells) obtained by fusion with tumor cells may also be used. In particular, methods using mice as mammals are often used. For example, mouse (Balb/
c) using 1α,25-dihydroxyvitamin D3
The amino acid derivative-carrier protein binding antigen was dissolved in a buffer solution or physiological saline, and the same amount of Complete Freund's Adjuvant was added to mix and suspend the emulsion, which was then administered subcutaneously to mice, and then administered approximately every week. multiple additional doses,
Perform sensitization. Thereafter, cell fusion is performed 3 to 5 days after the final sensitization. Cell fusion uses antibody-producing progenitor cells in the lungs 3 to 5 days after the final sensitization. This is because it is said that lung cells are most likely to be obtained, and lung cells are usually used for fusion around 3 days after the final sensitization, when the number of such cells is highest. After ~5 days, 1α, 25
- Lung cells, which are dihydroxyvitamin D3 antibody-producing cells, are collected and fused with established myeloma cell lines that can be cultured for a long period of time. The established cell line that can be cultured for a long period of time may be any cell that produces immunoglobulin or its related proteins that grows and proliferates for a long period of time under in vitro or in vivo conditions, and tumor cell lines that proliferate vigorously are usually used. Preferred typical examples include myeloma cell lines (myeloma cells) P3-NSI/1-Ag4-1 and P3-X63-A.
g8UI, SP2/U-Ag 14, MPCII-45
, 6°7G, and 1.7, among which P3-X63-Ag8U1 is preferred in the present invention. These cell lines are cultured and preserved in a normal cell culture medium.

For example, culture is performed in a medium containing 10% FC3-supplemented RPM11640 (product name: RPM11640: manufactured by Flor Laboratory) with addition of glutamine, pyruvate, penicillin, streptomycin, etc., and storage is performed in a culture medium containing the same medium supplemented with S-azaguanine. is used. In cell fusion, 1 to 3×10 8 myeloma cells may be used for one fusion. Lung cells, which are antibody-producing cells used for fusion, are obtained by dissecting the mouse after sensitization as described above, taking out the lungs, cutting them into pieces, and grinding them on a cell mesh to obtain a lung cell suspension. Prepare. Cells are washed and used for fusion with myeloma cells, but 1
Usually, 1 to 3 x 10" cells are used for the second fusion. Cell fusion is performed by mixing the lung cells and myeloma cells obtained as above. The mixing ratio is determined empirically. Myeloma cells:lung cells are used at a ratio of 1:3 to 10. Cell fusion is performed in a hybridoma medium.

For fusion, it goes without saying that it is preferable to use a conventional cell fusion method, ie, using a fusion promoter such as Sendai virus or polyethylene glycol (PEG), but in the present invention, the use of PEG is optimal. The cells thus fused with the addition of a cell promoter are dispensed into the above-described hybridoma medium and cultured. In this way, the fusion of mouse lung cells and mouse myeloma m follicles takes place.
In order to select only fused cells, the fused cells are selectively grown in HAT medium. HAT medium is a medium obtained by adding hyboxanthin, aminopterin, and thymidine to the above-described hybridoma medium. Generally, two or more hybridomas selected in this way grow per hole in the cell selection plate, and there is a possibility that two or more types of antibodies are secreted, and there are other hybridomas that cannot secrete antibodies. Since they may be mixed together, it is necessary to separate individual clones in order to obtain cells with the same properties. That is, it is necessary to perform cloning. As a cloning method, the limiting dilution method and the soft agar method are used, but the limiting dilution method is convenient in the present invention.

If you want to preserve the hybridoma that secretes a high titer of 1α,25-dihydroxyvitamin D3 antibody obtained in this way, it is best to freeze it as early as possible. For example, put the cell solution into a small freezing test tube or ampoule, freeze it in a -80°C freezer, and store it in liquid nitrogen.Furthermore, as an example of the hybridoma proliferation method, is preset with pristane (pristan+2+ 6+ 1
0+ 14-tetramethylpentadeca
Antibodies can also be prepared in large quantities by administering the above-mentioned hybridoma into the peritoneal cavity of pristane-treated mice that have been intraperitoneally administered with ne, Aldrich vJA), and collecting ascitic fluid about 10 days later. The hybridoma may also be cultured in RPMI medium containing fetal bovine serum, Dulbecco's modified Eagle's medium, or the like. The obtained antibody is purified by commonly used purification means, such as ammonium sulfate fractionation, followed by ion exchange chromatography, gel filtration, affinity chromatography, etc., to perform IgG fractionation.

The purified 1α,25-dihydroxyvitamin D3 monoclonal antibody can be obtained in this way. Also, 1
α,25-dihydroxyvitamin D3 monoclonal antibody-producing cells may be grown as tumors in animals that share the same histocompatibility antigen or athymic nude mice, and then collected and purified.

Furthermore, when quantifying 1α,25-dihydroxyvitamin D3, 1α,25-dihydroxyvitamin D3 polyclonal antibody or 1α,25-dihydroxyvitamin D3 monoclonal antibody (hereinafter collectively referred to as 1α225-
Dihydroxyvitamin D (sometimes abbreviated as antibody) is
It may be used as it is in a soluble state, or it may be used as a solid phase substance immobilized on an insoluble carrier. As such a solid phase, an insoluble carrier and lα,25-dihydroxyvitamin D3 antibody are bound together using a polyfunctional reagent, and this bound antibody has antibody activity against lα,25-dihydroxyvitamin D3. All you have to do is keep it.

The polyfunctional reagents used include amino groups, water, 1. H
Any polyfunctional reagent may be used as long as it has two or more groups capable of reacting with functional groups such as , carboxyl groups, and thiol groups. acid, dicarboxylic acid such as adipic acid or its reactive derivative, diisocyanate compound such as hexamethylene diisocyanate, 2,4-toluene diisocyanate, diisothiocyanate compound such as hexamethylene diisothiocyanate, maleimide Maleimidocarboxylic acids or reactive derivatives thereof such as benzoic acid, maleimidophenylacetic acid, N,N'-ethylene bismaleimide, N,N''
-〇-Shimaleimide compounds such as phenylene dimaleimide, bisdiazobenzidine, diethylmalonimidate,
Thiocarboxylic acid compounds or reactive derivatives thereof such as dimethyladipineimidate, N,N'-polymethylenebiiodoacetamide, 3-(2''-benzothiazolyl-dithio)propionic acid, 3-(2'-pyridyldithio)propionic acid These polyfunctional reagents include 1α.

25-dihydroxyvitamin D: amino group of l antibody.
It may be selected in consideration of bonding with a functional group such as a carboxyl group, a hydroxyl group, or a thiol group. The insoluble carrier used may be one that has a group capable of reacting with a functional group other than the functional group of the multifunctional reagent to which the antibody can be bound, such as insoluble carriers such as albumin and gelatin. In order to insolubilize proteins, agarose, cellulose, dextrin, etc. by epichlorohydrin treatment, cyanogen bromide treatment, and amino group insertion, a corresponding spacer is introduced and insolubilized acrylonitrile, acrylic acid, and acrylic acid. ester,
Insoluble synthetic polymeric carriers such as polymers or copolymers of methacrylic acid, methacrylic acid esters, vinyl alcohol, vinyl acetate, styrene, aminostyrene, chlorostyrene, maleic acid, fumaric acid, silicon, aluminum, etc. Examples include insoluble inorganic carriers in which functional groups such as amino groups are introduced into inorganic compounds. Furthermore, the insoluble carrier used may be one that can bind 1α,25-dihydroxyvitamin D and the antibody through mere physical adsorption.

These insoluble carriers usually have a particle size that can be easily isolated by means such as filtration, for example, a diameter of 1 m.
The diameter is preferably 5 mm or more, and bead-shaped ones are often used. Further, instead of being bead-shaped, a spindle-shaped tube similar to the shape of the bottom of an antigen-antibody reaction tube may be used.

When introducing a reactive group into the 1α,25-dihydroxyvitamin D3 antibody using a spacer introduction reagent, for example, dialdehyde compounds such as succinaldehyde, glutaraldehyde, and adibaldehyde, ω-aminobutyric acid, ω- Reactive derivatives of aminoglutamic acid such as acid chloride, succinimide ester, and p-nitrophenyl ester; reactivity of dicarboxylic acid compounds such as malonic acid, succinic acid, glutaric acid, and adipic acid; i-form, hexamethylene diamine, deca Diamine compounds such as methylene diamine, reactive derivatives such as 3-(2'-pyridyl-dithio)propionic acid, 3-(2''-benzothiazolyldithio)propionic acid, S-acetylmercaptosuccinic anhydride, 1 of spacer introduction reagents such as thiol compounds such as 2-aminoethanethiol
Four new functional groups such as an aldehyde group, a carboxyl group, an amino group, and a thiol group may be created by using one or more of these groups.

The reactive groups of such an insoluble carrier are then added to 1α,
The 25-dihydroxyvitamin D3 antibody is attached directly or using a polyfunctional reagent, and in the condensation, prt6. Each reaction may be carried out at 0 to 40°C in a buffer solution, an organic solvent, or a mixed medium thereof at a temperature of 0 to 8°C. As another method for producing a solid phase material, adsorption and immobilization may be performed by utilizing the physical adsorption ability of the insoluble carrier used. Furthermore, a second antibody obtained by immunizing other species of animals, especially large mammals, with the normal immunoglobulin fraction present in the serum of the animal used for antibody production of 1α,25-dihydroxyvitamin D. Using an antibody, this second antibody is immobilized on an insoluble carrier, and then 1α,25-dihydroxyvitamin D and the antibody are bound to this by an antigen-antibody reaction.
- Dihydroxyvitamin D3 The homolog may be obtained by immobilization means that hardly deactivate the antibody activity. Furthermore, the homologue obtained in this way may be washed and stored.

Furthermore, instead of using the solid phase described above, 1α, 25-
When quantifying 1α,25-dihydroxyvitamin D3 in a test solution using a dihydroxyvitamin D3 antibody in its soluble state, a certain amount of iodine radioisotope-labeled 1α expressed by the formula (Tar) must be used in advance. , the above labeled 1α, 25-dihydroxyvitamin D3 derivative and the test solution produced by adding an optimal amount of 1α, 25-dihydroxyvitamin D3 antibody to the test solution to which the 25-dihydroxyvitamin D3 derivative has been added. 1 existing in
α,25-dihydroxyvitamin D3 and lα,25-dihydroxyvitamin D3, unreacted labeled 1α,25-dihydroxyvitamin 03 with antigen-antibody reaction conjugate with antibody
A specific antibody against 1α,25-dihydroxyvitamin D is used to separate the 3M conductor.

This specific antibody is also called a second antibody, and the second antibody uses, as an antigen, a normal immunoglobulin fraction present in the serum of the animal used to produce the 1α,25-dihydroxyvitamin D3 antibody. Antiserum can be obtained by immunizing other species of animals, especially large mammals, by subcutaneous or subcutaneous injection. This second antibody may be obtained from antiserum by known purification means if necessary, and it is convenient to use it in the form of antiserum.

Furthermore, the 1α,25-dihydroxyvitamin D3 antibody obtained as described above and the iodine radioisotope-labeled 1α,
A method for quantifying 1α,25-dihydroxyvitamin D3 in a test solution using a 25-dihydroxyvitamin D3 derivative will be described. First, 1α,25-dihydroxyvitamin D,
In a test liquid, such as human serum, whose content is to be measured, first, 1α,25-dihydroxyvitamin D3 in the serum is extracted. Add the same amount of organic solvent as the serum, stir and leave to stand, extract by centrifugation, separate the obtained supernatant, and roughly purify 1α,25-dihydroxyvitamin D3 by column chromatography. , 25-dihydroxyvitamin D3 elution fraction is collected. More preferably, it is purified by charging it onto an HPLC column and eluting it. The eluted fraction of 1α,25-dihydroxyvitamin D was confirmed in advance using tritium [3H] radioisotope-labeled lα,25-dihydroxyvitamin D3, and both fractions were collected to obtain 1α,25-dihydroxyvitamin D.
- Recover dihydroxyvitamin D3. This 1α, 2
The 5-dihydroxyvitamin D3-8 fraction was dried under reduced pressure, replaced with argon gas, and further dissolved in ethanol.
Used for measurement as a test solution. Next, 1α in the test solution
, quantifying 25-dihydroxyvitamin D3. first,
A certain appropriate amount of iodine radioisotope labeled 1α is added to the test solution.
, 25-dihydroxyvitamin D, and derivatives are added, then an optimal amount of 1α,25-dihydroxyvitamin D3 antibody' is added, and the mixture is heated to about 4 to 5°C in a solvent for antigen-antibody reaction, such as phosphate buffer or veronal buffer. Approximately 15 hours to 7
The mixture is incubated for 2 hours to induce a competitive reaction between the radioactive iodine-labeled hapten and the unlabeled hapten to be quantified against the antibody. Thereafter, as a result of a competitive reaction between the radioactive iodine-labeled hapten against the antibody and the unlabeled hapten to be quantified, the generated antigen-antibody conjugate, particularly iodine radioactive isotope-labeled 25-dihydroxyvitamin D, derivative = Iα
, 25-dihydroxyvitamin D, antibody conjugate (B)
and the unreacted i-isomer (F) remaining without participating in conjugate formation, especially the radioisotope-labeled iodine 1α,25-hydroxyvitamin D3 derivative, which is the unreacted ifl Lu-form. To do this, filter or centrifuge '4 (30QQ
rpm, 15 minutes) to measure each radioactivity, calculate the B/(I3+F) or B/F radioactivity, and calculate the 1α,25-dihydroxyvitamin D3 in the test solution. Find the amount of (H). That is,
As the amount of H increases, the radioactivity of B decreases and the radioactivity of F increases.

Therefore, by measuring the radioactivity of B and F under conditions where an unknown amount of H is present, the unknown amount of H can be determined from a standard curve prepared in advance using known amounts.

In addition, in B-F separation, when the antibody used remains soluble and a two-antibody method using a second antibody is adopted, after a competitive reaction, the second antibody, more preferably an IgG containing the second antibody, is used. , lα as a carrier if necessary
, 25-dihydroxyvitamin D, and IgG from the same species of animal used for antibody production were added and incubated for 1 to 12 hours.Then, the conjugate produced by the antigen-antibody reaction was centrifuged at 300 rpm for about 10 to 30 minutes. The precipitated precipitate portion (B) and its supernatant portion (F) may be separated, and the radioactivity of either the washed precipitate or the supernatant may be measured.

By this measurement, 2 pg/Test ~2
A standard curve up to 56 pg/Test can be created,
In this measurement system, the reaction time is 16 hours at 5°C or 1 hour at 37°C, which is rapid, the post-reaction processing operation is simple, and the dilution of the test solution and addition recovery test also demonstrate linearity. Therefore, the accuracy is quite high.

Moreover, even if conventionally used DBP is used instead of the antibody, the lα,25-dihydroxyvitamin D,-iodo [l! s■] Reacts with radioisotope-labeled derivatives, and a standard curve can be created between 1 and 32 pg/'l''est. Obtained from the serum of

Next, the present invention will be specifically described with reference to Examples and Reference Examples, but the present invention is not limited thereto.

Reference Example 1 Extraction and purification method for 1α, 25- (OHz > Dff in serum) 0.5 mA of acetonitrile for 0.5 mA of serum!
was added, stirred using a Poldex mixer, and then allowed to stand for 30 minutes. Thereafter, the mixture was centrifuged (3000 rpm, 10 minutes), and 0.8 mff1 of the resulting supernatant was collected. 0.8 ml of the supernatant was added to a Sesoppasok C-18 cartridge column (trade name, formerly LLIPORE).

(manufactured by Waters) and was roughly purified by elution with aqueous acetonitrile. At that time, Cefdepack C-18
The cartridge column was activated with ethanol and equilibrated with 50% acetonitrile according to a conventional method.

After charging, wash with 4 ml of 50% acetonitrile, then elute with 4 ml of 64% acetonitrile (1 cx, 2
5- (OHg) both parts including Dff), then 73
% acetonitrile (both fractions containing 25-hydroxyvitamin D and 24.25-dihydroxyvitamin D).

The 64% acetonitrile elution fraction was dried under reduced pressure and replaced with argon gas to obtain 1α,25-(OH)zD.
j fraction was obtained. More preferably, this crudely purified fraction is
-Dissolved in 200 μl of a mixed solvent of hexane-isoprobanol (9:1) and analyzed by HPLC Zorba.
x -S I L (Dupon L Company V) O, 46X2
Purification was performed by charging a 5 cm column and discharging. Here, 1α, 25-
(OII) The elution fraction of Z D3 was pre-contained with tritium (
'11) label lα, 2s-(orJ)z D3 (
26,27-methyl-3H). H.P.L.
The lα, 25- (on) 2 Dff elution fraction (Rt = 9 to 13 minutes) of C was dried under reduced pressure and replaced with argon gas. This elution fraction was dissolved in 10 μβ of ethanol and used for measurement.

The recovery rate of 1α,25-(on)2D3 by this operation was 95±2% (n=10).

Reference Example 2 Synthesis of 3-(N-fluorenylmethyloxycarbonyl)aminopropionic acid! 1-(9-Fluorenylmethyloxycarbonyloxy)succinimide (674mg, 2m mole)
is tetrahydrofuran [hereinafter referred to as T)IF]-dimethylformamide [hereinafter referred to as DMF]-HzO(1
: 2 : 2) Dissolve in a mixed solvent of 25 mA and add γ-amino-n-propionic acid (also known as β-alanine) (178
mg, 2 mmole) and reacted at room temperature overnight. Thereafter, the reaction solution was distilled off under reduced pressure, and the residue was transferred to a silica gel column [packing material: Wakogel C-200,7
5g; Elution solvent CHCl, -methanol-9:1]
The product was separated and purified to obtain 538.8 mg (yield: 86.5%) of the title target product.

NMR, δ(DMSO-d6) 2.50-2.60 (2n, t, -CIlz-C
O) 3.28J, 35 (211,m, -CHz-N
)4.33-4.44 (311,m, Fmoc
)7.35-8.06 (811,m, Fmoc
) Example 1 1α,25-dihydroxyvitamin D3 ((3-(N-
Fluorenylmethyloxycarbonyl)amino)propionic acid ester] [Hereinafter referred to as 1α.

Synthesis method of 3-(N-fluorenylmethyloxycarbonyl)aminopropionic acid (hereinafter referred to as Fcom-β-Ala)
7.775 mg (0,025 mmol e) in 2 ml of anhydrous tetrahydrofuran (THF)! Trimedyl acetyl chloride (also known as pivaloyl chloride) is dissolved in
．． 07μN (0,025m mole) and dimethylaminopyridine (hereinafter referred to as DMAP) 3.05
mg (0,025 mmole) was added while maintaining the temperature at -15°C, and the temperature was maintained at -15°C for about 10 minutes. after that,
1α,25-dihydroxyvitamin D3 10.4mg
(0,025 m mole) and incubated at -15°C for 5
minutes and then kept at 0° C. for 1.5 hours. After the reaction, 0.5 ml of methanol was added, and the reaction solvent was evaporated under reduced pressure, and subjected to silica gel column chromatography [Filling product name: Wakogel C-200, 150 g; elution solvent: ethyl acetate.
The mixture was separated using hexane (4:l) to obtain the three components shown below.

Fl: lα, 25 (OH)z D3 bis
(β-Ala-Fmoc) Abbreviation for (lα, 25-dihydroxyvitamin D, -1α, 3β-O-bis((3-(N-fluorenylmethyloxycarbonyl)amino))propionic ester] Rate: 19.1% (Rf: 0.59) F2: lα, 25- (Off) 2D, -3β-0-β-A
la-Fmoc (abbreviation for lα, 25-dihydroxyvitamin D3-3β-0-((3-(N-fluorenylmethyloxycarbonyl)amino)propionic acid ester)) and 1α, 25- (OH)2D3 1α O -β-A
la-Fmoc (1α.

25-dihydroxyvitamin D3 1α-0-[(3-
(N-fluorenylmethyloxycarbonyl)amino)
Mixture yield of propionic acid ester]: 20.3% (Rf: 0.47) F3: Recovery yield of raw material 1α, 25-(OH)z Ds: 4
1.2% (Rf: 0.20) obtained F2 fraction (1α
, 25 (011)'z Dz-3β-0-β-
Ala-Fmoc and lα,25- (OTT)
zDi-1cx-0-β-Ala-Fmoc mixture) was analyzed using HPLC (column brand name Zo r b
ax-ODS; φ0. 46X25cm
;; Moriyama solvent 10% 1120-methanol; flow rate 1 m
1/min) into the respective components.

The physical properties of each product are shown below.

lα, 25-(01() Z Di -3β-O-
β-Ala-Fmoc HPLCRt=17.46 min (conditions are the same as above) NMR, δ (CDCj!:+)I) pmo,
55 (3H, s), 0.94 (3H, d),
1.22 (611,s), 2.53 (2H,
t), 3.44-3.48 (311, m), 4.
22(IH,t), 4.37-4. 41 (3
1-T.

m), 5.03 (LH, d), 5.27~
5.30 (211, m), 5.3 5 (IH
, m), 6.01 (IH, d), 6. 34 (I
H.

d), 7. 29-7. 77 (811, m
) UVλ'4+'i''A (nm) 300.
s, 266.3214.6 1α, 25 (OH)z Di 1 α
-0-β-Aia-Fmoc HPLCRt=14.92 min (conditions are the same as above) NMR, δ, (CDC13) ppm0.50
(3H, s), 0.90 (31-T, d), 1.
20 (3H,s), 1.27 (3H,s),
4.04-4.05 (LH, m), 4.21
(LH, m), 4.41~4.46 (2H, m)
, 5.05 (IH, d) , 5.27 (LH,
t), 5. 32 (IH, m), 5.
50 (LH, t), 5.90 (IH, d
), 6.30 (LH, d), 7.28-7.79
(8H, m) UVλatop (nm)
300.4.266.4214.3 1 α, 25 (OH)z Di bi
s (β-Ala-Fmoc) NMR, δ (CDC7!:+) ppm0,
50 (3H, s), 0. 90 (3H, d
), 1, 20 (3H,s), 1. 26
(3H,s), 2, 40-2. 65 (
4H, m), 3. 30-3, 55 (411
,m) ,4. 10-4. 50 (6H, m)
,5. 05-5. 60 (6T[, m)
, 5, 90 (llr, d) , 6. 32
(IIr, d), 7, 20-7. 83
(16H,m) Example 2 (1) 1α,25-dihydroxyvitamin D3-3β-
0-(3-aminopropionic acid ester) [hereinafter referred to as 1α
, 25- (OH)z Di -3β-O-β-ΔIa
synthesis method lα, 25-dihydroxyvitamin D3-3β-O-(
(3' = (N-fluorenylmethyloxycarbonyl)amino)propionic acid ester] [Hereinafter, 1α, 2
5 (OH)z Di 3β-0-β-Aha
-Fmoc) 13.4mg (1B, 86X10
-'m mole) was dissolved in 2.5 mA of ethanol, and 2.5 mj2 of morpholine was added at room temperature. This was stirred for 2 hours at room temperature under an argon atmosphere, and the reaction solution was distilled off under reduced pressure to obtain a crude product of the target compound. In order to remove the morpholine remaining in this crude product, LH20 (trade name) (Hz 0, 10 ml capacity) was used,
Washed with 20-30 ml H2O. The target product was eluted with methanol, and Amp rep-NO3 (manufactured by Amerjam, 50 Qmg, hexane-dichloromethane = 1:
1) and hexane-dichloromethane (1:
1) 30mJ! The target compound was obtained by washing with water and eluting with ethanol.

Yield 8.73X10-'m mole (yield 46.25%) UV λEtOHH2O4,2Hm aX Ninhydrin coloration Positive TLC (Silica gel Merk 5715; developing solvent ethyl acetate-methanol-1:4) Rf: 0.15 (2) 1α,25-dihydroxyvitamin D3-1α-
0-(3-aminopropionic acid ester) [hereinafter referred to as 1
Synthesis method of α, 25 (0 ■ I) z Dt 1α-〇-β-Ala) In the above (1), 1α, 25- (011) Z D3
−3β−〇−β−A l a−Fmo c instead of 1
α, 25- (011)Z D3-14-Q-1β-A
The target compound was obtained in the same manner as in (1) except that la-Fmoc was used.

Yield 62.5% tOH Uv λmax: 265.4βm Ninhydrin coloration Intestinal T1. C (silica gel Mark 5715 ethyl acetate-meth/-l = 1:4) Rf: 0.25 Example 3 1α,25-dihydroxyvitamin D3-3β-0-(
3-(USA-amino)-propionic acid ester] and preparation of 1α,25-dihydroxyvitamin D antiserum (1) 1α,25-dihydroxyvitamin D3-3β-
0-[1-(BSA-amino)propionic acid ester]
Preparation of 1 obtained by deprotecting the F m oc group
α, 25-dihydroxyvitamin D, -3β-10-(
3-amino-propionic acid ester) (12,2 mg
, 25.05X10-'m mole) in THFlm
ff) 8 answers. Tris buffer (0, 1M, pF
48.6) 10mj! to I”3SA (MW.

Approximately 65000.32.5mg, 1150X25゜osx
to -3m mole) was dissolved, and after cooling to 0°C, 1
-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (6.3mg, 1.3X25.05X1
0-'m mole) and the inertia force was 11. This solution is 0
After reacting for 3 hours at °C, 10 m of chloroform was added to the reaction solution.
N was added four times to remove unreacted 1α,25-dihydroxyvitamin D3-3β-0-(3-aminopropionic acid ester). The aqueous solution layer was freeze-dried to obtain 28.0 mg of a freeze-dried product of the title target, with 1α, 25-dihydroxyvitamin D3-3β-0 per B5Al molecule.
18 molecules of -(3-aminopropionic acid ester) were bonded via the amino group of 3, and this was abbreviated as the formula of the title compound.

(2) Preparation of 1α,25-dihydroxyvitamin D3 antiserum Lyophilized product lα,25-dihydroxyvitamin D3-3β-0-(3-(USA-amino)-
Propionate ester] [hereinafter abbreviated as antigen] was dissolved in Tris buffer (0.1M, pH 8.6), and the same amount of Complete Freund's adjuvant was added thereto.
Sufficiently mix and suspend the antigen at 1 Mg to 100 μg/m
It was adjusted to be 1. This emulsion was injected subcutaneously into rabbits approximately 10 times at intervals of 2 weeks at an amount of IOT~2Q Or/animal. During this period, blood was collected midway every 10 days to check the antibody titer, and when the antibody titer reached the maximum, whole blood was collected, left to stand at room temperature for 60 minutes, allowed to solidify, and then spun at 3000 rpm for 10 minutes. Centrifuge for 1 min,
, 25-dihydroxyvitamin D.

Antiserum containing antibodies was obtained, fractionated with ammonium sulfate, and an IgG fraction was collected.

(3) Preparation of 1α,25-dihydroxyvitamin D3 monoclonal antibody B a Rb/c mice (female, 4 weeks old) were given (1)
The freeze-dried 1α,25-dihydroxyvitamin D,-3β-0-(3-(BSA-amino)-propionic acid ester) [hereinafter abbreviated as antigen] obtained in pI-I7
．． C, F, A
After one week, 50γ was administered subcutaneously again, and after two weeks, 50γ was administered intraperitoneally with the antigen alone. Three days after this final sensitization, mice were dissected and 3l1lI! The viscera was removed, chopped, and ground on a cell mesh to prepare a lung cell suspension. Cell fusion is mouse myeloma cell P3-X-3-Ag8
It was carried out according to a conventional method using U1. First, 30%
A PEG (molecular weight 1000) aqueous solution was incubated at 37°C, and then the above lung cells and myeloma cells (total number of both cells, 4 x 10' cells, 5:l) were suspended in RPMI-only medium (5 rrW), and both cells The cells were combined and mixed slowly, centrifuged at 10QQrpm for 10 minutes, and the supernatant was removed by aspiration.The cells were washed again with RPMr medium (10mff), and after centrifugation, the supernatant was removed by aspiration.The test tube was gently tapped. Mix the cell pellets and add l, Qm, i
! PEG solution was slowly added and gently stirred. After incubation at 37°C with gentle shaking, the fusion reaction was stopped by slowly adding regular medium (IQm#) and the cells were suspended again.

This was centrifuged at 000 rpm for 5 minutes, and the precipitated cell pellet was dispersed by gently tapping the test tube, then gently suspended in IT A T medium (5 ml), then HA T medium (1 m#). The cell suspension was transferred to a 96-well plate and microscopically examined for live II cells.
Cells were grown in a CO2 incubator at 200μ/well. After selecting the fused cells in HAT medium, they were cloned by limiting dilution. Using the obtained clone suspension, Q, 5mj! Pristane treatment Ba
1b/c mice were allowed to proliferate intraperitoneally, and ascites and fluid were collected from the mice. The TgC fraction was collected by a conventional method, and the TgG fraction was further purified by affinity chromatography using protein A-bound Sepharose CL-4B. ,
This was used as a monoclonal antibody. The subclass of IgG obtained from this was IgG1.

Example 4 Radioisotope labeling of (11 iodine (+25 r) 1
α.

25-dihydroxyvitamin D3 derivative 1α, 25-dihydroxyvitamin D3-3β-O-[(3-(N-3
-(4-Hydroxy-3-iodo(1251)phenyl)propionyl)aminocopropionic acid ester production method 1α,25-dihydroxyvitamin D3-3β-0-(3- aminopropionic acid ester) (72,8n mole/25μj
Add tetrahydrofuran (2EtOH) to an ethanol solution of
THF) 75μ/, ) ethylamine (Et3N
) 300p mole and iodine [125)) Porton-Hunter reagent (NEN, NEX-120-10,22
00Ci/m mole, 0.33mC1/100μ
#Benzene: 50μCi) was added and heated at room temperature for 24 hours under aerobic atmosphere.
Allowed time to react. This reaction solution was separated and purified by HPLC to obtain the title target product.

Radioactivity recovery rate 27.4 μCi (54.7%) HP
L C conditions ■ Column Zorbax-ODS; φ4.6x250mm
5 μm Elution solvent 15% ■ 1 □ 0-methanol; flow rate 1 mn/min; Rt: 13.5-15.6 minutes ■ Column Zorbax-
3IL; φ4.6X250mm 5μm+Prec
olumnHφ4.6X50mm Elution solvent 20% isopropanol-hexane; flow rate 1m
1/min; Rt: 16-22 min (2) 1α,25-dihydroxyvitamin D3-1α-0-[(3-(N-3-(
Method for producing 4-hydroxy-3-iodo(1251)phenyl)propionylaminocopropionic acid ester In the above (1), 1α, 25 (OH)2 D:+
-3β-0-β-Ala instead of 1α, 25-(O
H) 2 D3-1 αO-β-Ala was used (
The desired compound was obtained in the same manner as in 1).

Radioactivity recovery rate 30.2 μCi (60.4%) HP
LC ■ Conditions are the same as above ■, Rt: 14°2 ~ 16.8
Minute ■ Conditions are the same as above ■, Rt: 13° 6-16.1 minutes Example 5 (1) 1α,25-dihydroxyvitamin D3-(3β
-〇- and -1α-0)-(3-aminopropionic acid ester) mixture synthesis method 1α,25-dihydroxyvitamin D3-[(3-(N
-fluorenylmethyloxycarbonyl)amino)propionic acid ester mixture 8.4 mg (11,83
X10-3m mole) Dissolved in 2m ethanol It, morpholine 2m7 at room temperature! was added and reacted for 2 hours at room temperature under argon atmosphere.

The reaction solution was distilled off under reduced pressure, and the residue was crudely purified using LH-20 (trade name) (H20, 10ml (manufactured by 7 Almasia) in the same manner as in Example 3(1), and then further purified with Am as in Example 3(1).
Purified with prep-NHz (500 mg, manufactured by Amerjam).

Recovery rate 5.13×lO-'m mole (43
゜4%) (1α,25-dihydroxyvitamin D3-3β-〇-(3-aminopropionic acid ester) (hereinafter referred to as -3β-0-) and 1α,25-dihydroxyvitamin D3-1α-0- (as a mixture containing 3-aminopropionic acid ester) (hereinafter referred to as -1α-0-) in a ratio of about 2.5:1) UV A”m
Grain 265. Onm ninhydrin coloring positive TLC (silica gel Merck 5715 developing solvent ethyl acetate-methanol-1:4) R' (3β-0-):
Q, 15 Rf: (1α-0-):0125 (2) Iodine [I2S■] radioisotope labeled 1α.

25-dihydroxyvitamin D, -(3β-〇- and l
α-0-)-[(3-(N-3-(4-hydroxy-3
-Production method of iodo(125I)phenyl)propionyl)aminocopropionic acid ester and separation/purification method of the mixture Ethanol solution of the mixture obtained in (1) above (205 nmole/10
0 II l ethanol) and THF30. crjl! ,
) ethylamine 600pm.

le and iodine (Its l )-Porton-Hunter reagent (NEN, NEX-120-10, 2200C4/
m mole, 0.33 mC1/100 μl benzene; 100 μL benzene) was added, and the mixture was reacted for 24 hours at room temperature under an argon atmosphere. The reaction solution was purified by HPLC to obtain the title target product as a mixture.

Radioactivity recovery rate 50.7 μCI (50.7%) HP
L C Conditions are the same as above, Rt: 13-17 minutes ■ Conditions are the same as above, Rt: 13-2 2 minutes Furthermore, the reaction solution or the purified mixture is subjected to HPLC
3β-0- and 1α-0- were separated and purified by precision fractionation under the conditions (2).

1α-0-Rt:13. O ~ 15.6 minutes Radioactivity recovery amount
16.3 μCi 3β-0-Rt: 15. Q~21.2 minute radioactivity recovery amount
The recovery ratio of 3465 μCi 1α-O- and 3β-0- was 1:2.1,
lα-O- and 3β-0- in the raw material before F m oc removal
The ratio of existence was one defeat.

Example 6 Preparation of standard curve by radioimmunoassay for 1α, 25 (OH) 2 Ds An ethanol solution of a standard product of 1 α, 25 (OH) 2 H3 was diluted and used as a standard solution of 0 (for Bo), 204
8 (for NSB), 4.8.16.32.64.128
．． Make an ethanol solution of 256.512.11024p/20μβ and dispense 20μβ into two tubes for each concentration. Next, add 2 ethanol solutions of the obtained iodine (IZS)-labeled compound (3β-〇-) to each tube.
Dispense 0 μβ (approximately 12,000 cpm). moreover,
Antiblood obtained by immunizing sheep? p
Add 1 ml of the solution diluted in H8,6 Tris buffer to each tube and incubate at 5°C for approximately 16-24 hours. Add 200 μl of dextran-charcoal to this reaction solution, and centrifuge at 5°C for 30 minutes to
F separation was performed. 1 ml of the supernatant was collected and measured using an Autowell γ-counter. Calculate the average of each concentration of the obtained counts, subtract NSB from that value (B), and plot the value calculated from the following calculation formula [3-NSB (X 100) to obtain the 1α, 25(OH
)2 A standard curve of Dt is obtained.

〔Effect of the invention〕

As described above, the present invention provides lα labeled with radioactive iodine, which has high radiation energy and is excellent in versatility.
25-dihydroxyvitamin D3 derivatives can be provided. Conventionally, there have been various vitamin D3-labeled compounds using tritium ('H), which is a β-ray emitting nuclide with low radiant energy per unit time. Iodine [125
The vitamin Dz derivative labeled with [I] is thought to induce radical reactions and self-decomposition due to the instability of the conjugated triene structure, which is also a characteristic of vitamin D, and has not been reported to date. However, the iodine (1g5 1)-labeled 1α,25-
It has been found that dihydroxyvitamin D3 derivatives do not undergo structural decomposition due to gamma rays, are very stable, and can be used in radioimmunoassays.

In general, vitamin D3 is metabolized by the liver or kidney in vivo to become active vitamin D3. These active vitamin D3 types (25-hydroxyvitamin D3.1α, 25-dihydroxyvitamin D3), 1α-hydroxyvitamin D3.1α, 24-dihydroxyvitamin D3, etc. have been clinically shown to be effective against bone loss. It is used as a treatment for rough R syndrome, osteomalacia, etc.
Or it is being developed. The name of these drugs
Bed doses are usually significantly lower due to their strong physiological effects. On the other hand, pharmacological action has a correlation with drug concentration in blood/serum and tissue, and measurement of serum drug concentration in humans has particularly important clinical significance. Therefore, the present invention has high utility value in clinically measuring active vitamin D3s.

[Brief explanation of drawings]

The drawing shows radioactive isotope labeling of iodine (+25■) 1α
, a standard curve using 25-dihydroxyvitamin D3 derivatives. January 1st, 1990

Claims (7)

[Claims] (1) Formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, one of R_1 and R_2 is a hydroxyl group and the other is -O-CO-R_3-NH_2, and R_3 is an alkylene having 1 to 10 carbon atoms. A novel 1α represented by ]
, 25-dihydroxyvitamin D_3 amino acid derivative. (2) Formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, one of R_1 and R_2 is a hydroxyl group and the other is -O-CO-R_3-NH-R_4, and R_3 has 1 to 10 carbon atoms. 1α,25-dihydroxyvitamin D represented by an alkylene group, R_4 represents an amino protecting group]
There are formulas, mathematical formulas, chemical formulas, tables, etc. that are characterized by removing the amino protecting group from the _3 derivative in an inert solvent in the presence of a base. [In the formula, one of R_1 and R_2 is a hydroxyl group. The other is -O-CO-R_3-NH_2, and R_3 is an alkylene group having 1 to 10 carbon atoms.]
, 25-dihydroxyvitamin D_3 A method for producing an amino acid derivative. (3) The production method according to claim 2, wherein the amino protecting group is a 9-fluorenylmethyloxycarbonyl group. (4) Formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, one of R_1 and R_2 is a hydroxyl group and the other is -O-CO-R_3-NH-R_5, and R_3 has 1 to 10 carbon atoms. 1α,25-dihydroxyvitamin D_ labeled with an iodine radioisotope, represented by an alkylene group of
3 derivatives. (5) The radioactive isotope label of iodine 1α,2 according to claim 4, wherein the radioactive isotope of iodine is ^1^2^5I.
5-dihydroxyvitamin D_3 derivative. (6) The residue with the radioactive isotope of iodine is 3-(
4-hydroxy-3-iodo[^1^2^5I]phenyl)propionyl group or 3-(3,5-jodo[^1
The radioactive isotope labeling 1 of iodine according to claim 4, which is a ^2^5I]-4-hydroxyphenyl)propionyl group.
α,25-dihydroxyvitamin D_3 derivative. (7) The test solution has a formula ▲ a mathematical formula, a chemical formula, a table, etc. Iodine radioisotope labeling 1α,25-dihydroxyvitamin D_
3 derivative and 1α,25-dihydroxyvitamin D_3 antibody or vitamin D binding protein (DBP)
) Radioisotope-labeled 1α,25-dihydroxyvitamin D_3 derivative of the iodine produced by reacting with
1α,25-dihydroxyvitamin D_3 antibody conjugate or radioisotope labeling of the iodine 1α,25-dihydroxyvitamin D_3 derivative-radioisotope labeling of the iodine that has not reacted with the vitamin D binding protein conjugate 1α,25-dihydroxyvitamin D_3 1. A method for quantifying 1α,25-dihydroxyvitamin D_3 in the test solution, which comprises separating the derivative and then quantifying the amount of labeled iodine, which is one of the separated radioactive isotopes.