JPH0727763A - Antibody to 1alpha, 25(oh)2 vitamin d3 and its use - Google Patents

Abstract

PURPOSE:To obtain an antibody measurable with a sensitivity which is nearly equal to that of a radioreceptor assay by a method wherein bovine serum- albumin is bonded to a hemiglutaryloxy group to a hapten derivative by an N-hydroxysuccineimide method. CONSTITUTION:A hapten carrier protein complex obtained in such a way that a hapten derivative is synthesized and that bovine serum albumin(BSA) is bonded to a hemiglutaryloxy group to the hapten derivative by an N- hydroxysuccineimide method is used as an immunity source, it immunizes a lagomorpha, and a 1alpha, 25(OH)2 vitamin D3 antibody is obtained. The affinity constant of the obtained antibody is at 0.34 to 3.3X10<10>(M<-1>), its crossing-over reactivity with reference to other vitamin derivatives is at 6 & or lower when the reactivity with reference to 1alpha, 25(OH)2 vitamin D3 is set at 100 %. As the immunity source used to prepare the antibody, a source in which carrier protein such as the BSA or the like has been bonded to the tip of a bridge introduced into the 11alpha position of the 1alpha, 25(OH)2 vitamin D3 is used.

Description

Detailed Description of the Invention

[0001]

The present invention relates to 1α, 25 (OH) ₂
Antibodies against vitamin D ₃ , 1α, 25 using the antibodies
The present invention relates to a method for measuring (OH) ₂ vitamin D ₃ and a kit.

[0002]

2. Description of the Related Art Vitamin D₃Is photochemically synthesized in the skin
Alternatively, after being ingested from food, 25 (OH)
Tamin D₃Is metabolized to and transferred to the kidney, 1α, 25
(OH) ₂Vitamin D₃In vivo (liver,
24,25 (OH) in kidney, small intestine, etc.₂Vitamin D₃Na
Which are converted into various metabolites. Blood of these various metabolites
Accurate measurement of levels is important for clinical and nutritional diagnosis.
It is important.

Currently, as a method for measuring these various metabolites,
Is, for example, 25 (OH) vitamin D ₃, 24, 25
(OH)₂Vitamin D₃About Serum Vitamin D Binding
Competitive prot using protein
ein binding assay is 1α, 25
(OH)₂Vitamin D₃For use the receptor
Radio Receptor Assays Dominate Mainstream
It However, these methods require complicated preparation of biological samples.
Processing is indispensable, and there is a problem in sample processing capacity.

[0004]

Recently, an immunoassay represented by a radioimmunoassay is expected as a new methodology, and attempts have been made to produce antibodies against vitamin D ₃ and its various metabolites. However, conventional antibodies have the disadvantage that they cannot retain sufficient specificity because they are prepared from a hapten derivative having a carrier binding site on the 3-hydroxyl group or the 17-side side chain of vitamin D _3. It was

[0005]

Therefore, the present inventors have
Since the 11α-position of vitamin D ₃ is a site that is less likely to be metabolized in vivo and is present at a position sufficiently distant from both structures of the A ring and the 17-position side chain, this site is used as the binding site for the carrier protein. Assuming that an antibody having more excellent specificity than that of a conventional antibody can be obtained by using it, a novel hapten having a bridge at the 11α position was synthesized, and an attempt was made to prepare an antibody against this hapten.

That is, the following equation (1)

[Chemical 1]

The hapten derivative represented by
Immunize a rabbit with the obtained hapten-carrier protein complex in which bovine serum albumin (BSA) is bound to the hemiglutaryloxy group at the 11α position of the hapten derivative by the hydroxysuccinimide method. Have found that anti-1α, 25 (OH) ₂ vitamin D ₃ antibody can be produced. Then, this is further developed, and the molar ratio of the hapten derivative and BSA is 15 to
The present invention has been completed based on the finding that by controlling to 20 the target antibody with more excellent titer, affinity and specificity can be efficiently produced.

That is, the present invention relates to an antibody against 1α, 25 (OH) ₂ vitamin D ₃ having the following characteristics. Affinity constant (Ka value) 0.34 to 3.3 × 10 ¹⁰ (M ⁻¹ ) Cross-reactivity (%) 1α, 25 (OH) ₂ Reactivity to vitamin D ₃ is 1
The reactivity to other vitamin D ₃ derivatives when it is set to 00% is 6% or less. The present invention also relates to a method and kit for measuring 1α, 25 (OH) ₂ vitamin D ₃ using the above antibody. Hereinafter, the present invention will be described in detail.

(1) Antibody of the Present Invention The antibody of the present invention has at least the above-mentioned properties. The features of the antibody of the present invention will be described in more detail below. Affinity constant (Ka value) 0.34 to 3.3 × 10 ¹⁰ (M ⁻¹ ) Cross-reactivity (%) 1α, 25 (OH) ₂ Reactivity to vitamin D ₃ is 1
The reactivity with respect to other vitamin D ₃ derivatives when set to 00% is 6% or less, preferably 2% or less, more preferably 1% or less. Titer Optimal dilution ratio is 1300 times or more, preferably 18,000
~ 220,000 times. Measurement sensitivity The measurement limit in RIA is 2 to 10 pg / tube.

(2) Method for Preparing Antibody of the Present Invention As an immunogen used for preparing the antibody of the present invention,
It is important to use the one in which a carrier protein such as BSA is bound to the tip of the bridge introduced at the 11α position of 1α, 25 (OH) ₂ vitamin D ₃ . 1α, 25
A method for introducing a carboxyl group-containing bridge at the 11α-position of (OH) ₂ vitamin D ₃ , such as a hemiglutaryloxy group, is known (Abstracts of the 10th Symposium on Analytical Chemistry of Biological Components, Japan). 105-1
08, September 1992), the desired hapten derivative can be prepared according to this known method. The obtained hapten derivative and the carrier protein can be bound by the N-hydroxysuccinimide method. That is, by reacting a hapten derivative with N-hydroxysuccinimide in the presence of a water-soluble carbodiimide to introduce a succinimide group at the tip of a bridge of the hapten derivative, and reacting this with a carrier protein, the desired immunogen is obtained. Can be obtained.

As the carrier protein used for preparing the immunogen, there can be used proteins which are usually used in the immunogen of hapten such as BSA and keyhole limpet hemocyanin. The mol ratio of the hapten derivative per 1 mol of carrier protein is 15 to 20 (hapten / BSA = 15 to 20).
It is important to use the one. By using the immunogen having such a molar ratio, the antibody of the present invention can be efficiently obtained. The animal to which the immunogen is administered may be any of horses, sheep, goats, rats, mice, guinea pigs, dogs, pigs, rabbits, monkeys, pigeons, chickens, etc., particularly mice, rats, guinea pigs, rabbits, goats. Are convenient for use.

The administration of the immunogen to such animals may be carried out according to a conventional method, for example, various adjuvants such as complete Freund's adjuvant, incomplete Freund's adjuvant, alum adjuvant, aluminum hydroxide adjuvant, pertussis adjuvant and the above-mentioned adjuvants. An emulsion with the immunogen may be prepared and administered to the animal intravenously, intraperitoneally, subcutaneously or intradermally. The dose is preferably 0.01 to 10 mg / animal in the case of using rabbits and guinea pigs as animals, or about 0.001 to 1 mg / animal in the case of using mice and rats.

After the first administration, the same booster immunization as above is performed about 1 to 5 times every 1 to 4 weeks to induce antibody production against 1α, 25 (OH) ₂ vitamin D _{3 in} the animal body. Next, serum obtained by collecting blood from the antibody-induced animal and centrifuging it may be used as antiserum. If necessary, antibodies in blood may be purified by known methods (ammonium sulfate salting out method, ion exchange chromatography using anion exchanger such as DEAE cellulose, affinity chromatography using protein A-sepharose, molecular sieve chromatography, etc. The antibody of the present invention in serum can be isolated and purified by appropriately selecting or combining).

(3) Measuring method of the present invention The sample used for the measuring of the present invention is 1α, 25 (O
H) ₂ It is not particularly limited as long as it can contain vitamin D ₃ , and examples thereof include blood, serum, urine, body fluid, cultured cells or a culture solution thereof. The sample may be diluted with an appropriate buffer solution such as PBS before use for the measurement. The assay method of the present invention is characterized by using the antibody of the present invention as an antibody reagent. Therefore, the measuring principle and conditions adopted in the measuring method of the present invention are not particularly limited. For example, suitable methods and conditions for measuring 1α, 25 (OH) ₂ vitamin D ₃ may be appropriately selected and adopted from known methods described in the following documents.

Hiroshi Irie, "Continued Radioimmunoassay"
(Published on May 1, 1979 by Kodansha Co., Ltd.) Eiji Ishikawa et al. "Enzyme Immunoassay" (Second Edition) (Co., Ltd.)
Medical Institute, December 15, 1982) Clinical pathology Extra edition special issue No. 53 "immunoassay for clinical examination-technology and application-" (Clinical Pathology Press,
Published in 1983) "Biotechnology Encyclopedia" (CMC, Inc.)
(Published October 9, 1986) "Methods in ENZYMOLOGY V
ol. 70 "(Immunochemical tec
hnies (Part A)) "Methods in ENZYMOLOGY V
ol. 73 "(Immunochemical tec
hnies (Part B)) "Methods in ENZYMOLOGY V
ol. 74 "(Immunochemical tec
hnies (Part C)) "Methods in ENZYMOLOGY V
ol. 84 "(Immunochemical tec
hnies (Part D: Selected I
mmunoassay)) "Methods in ENZYMOLOGY V
ol. 92 "(Immunochemical tec
hnies (Part E: Monoclonal
Antibodies and General I
mmunoassay Methods)) [is published by Academic Press]

(4) Assay kit of the present invention The assay kit of the present invention comprises an antibody reagent attached to the kit.
And using the antibody of the present invention as described above.
Other reagents should be combined as appropriate according to the measurement method used.
You just need to match them. For example, the antibody used in the kit
Can be the antibody itself, but prevents non-specific adsorption
Therefore, it is preferable to use an active fragment of the antibody.
Good Active fragment of the antibody retains the characteristics of the antibody
What to do (eg, F (ab ') ₂, Fab ', Fa
b) various fragments]
You may. The preparation of these active fragments is
The body is protected by papain, pepsin, trypsin, etc.
Applying known methods such as limited decomposition method using
Can be performed (eg "immunobiochemistry (sequel
Chemistry Experiment Lecture 5) ", edited by The Biochemical Society of Japan, p. 89 (198)
6 years))).

When the antibody of the present invention is labeled and used, the labeling agent to be bound to the antibody is a radioisotope (eg ³² P, ³ H, ¹⁴ C, ¹²⁵ I etc.), an enzyme (eg β- Galactosidase, peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, catalase, glucose oxidase, lactate oxidase, alcohol oxidase, monoamine oxidase, etc., coenzyme / prosthetic group (for example, F
AD, FMN, ATP, biotin, heme, etc.), fluorescein derivatives (eg, fluorescein isothiocyanate, fluorescein ophthalvamil, etc.), rhodamine derivatives (eg, tetramethylrhodamine B isothiocyanate, etc.), umbelliferone and 1-
A fluorescent dye such as anilino-8-naphthalene sulfonic acid,
Luminol derivatives (for example, luminol, isoluminol, N- (6-aminohexyl) -N-ethylisoluminol, etc.) can be used. The binding between an antibody or its active fragment and a labeling agent is described in a publication [for example, "Sequel Biochemistry Experimental Course 5 Immunobiochemistry Research Method", Inc.
Tokyo Kagaku Dojin, (issued in 1986) 102-112
Page] may be carried out by appropriately selecting from known methods as described in the above.

Further, when the antibody of the present invention is used as a solid-phased antibody in which a solid phase is bound, examples of the material of the carrier substance for immobilizing the antibody include polyvinyl chloride, polystyrene, and threthin-divinylbenzene. Polymers, styrene-maleic anhydride copolymer, nylon, polyvinyl alcohol, polyacrylamide, polyacrylonitrile, polypropylene, synthetic organic polymer compounds such as polymethylene methacrylate, dextran derivatives (Sephadex, etc.), agarose gel (Sepharose, biogel) Etc.), polysaccharides such as cellulose (paper disk, filter paper, etc.), inorganic polymer compounds such as glass, silica gel, silicone, etc., and these are functional groups such as amino group, aminoalkyl group, carboxyl group, acyl group, hydroxyl group, etc. Base It may be the one you entered. The material of the carrier substance is preferably one having a low protein-binding ability, and as such a material, untreated polystyrene,
An example is polyvinyl chloride.

The shape of the carrier substance is flat (microtiter plate, disc, etc.), particulate (beads, etc.), tubular (test tube, etc.), fibrous, film-like, fine particle (latex particle, etc.), capsule-like. , Vesicles, etc. are exemplified, and a carrier having an appropriate shape can be selected according to the measuring method. In addition, liposomes (monolayer or multilayer lipid membrane) and the like can also be used as carrier substances for immobilizing antibodies. The binding between the antibody and the carrier substance is a known method such as a physical adsorption method, an ionic binding method, a covalent binding method or an encapsulation method (for example, “immobilized enzyme” (edited by Ichiro Chibata, Showa 5
March 20, 2000, published by Kodansha Co., Ltd.) can be adopted. Particularly, the physical adsorption method is preferable because it is simple. Further, the above bonding may be performed directly or may be performed between other substances through another substance.

[0020]

INDUSTRIAL APPLICABILITY The antibody of the present invention has a higher specificity for 1α, 25 (OH) ₂ vitamin D ₃ than previously reported antibodies. It is possible to measure 1α, 25 (OH) ₂ vitamin D ₃ in a sample with almost the same sensitivity.

[0021]

EXAMPLES The present invention will be described in detail below with reference to examples. Example 1: Preparation of antibody (1) Synthesis of hapten derivative Compound 3 (11
α, 25 (OH) ₂ cholesterol) (J. Chem.
Soc. , Perkin Trans. 1,1993,
The target hapten derivative (11α-hemiglutaryloxy-1α, 25 (OH) ₂ D ₃ ) represented by the formula (1) was prepared from 31) through 21 steps.

[0022]

[Chemical 2]

Reactions i) to xi shown in the synthesis flowchart
Details of the reaction condition of ii) are as follows. i) a: TBSCl, imidazole, DMF, room temperature,
b: Ac₂O, pyridine, room temperature, c: TBAF, TH
F, room temperature; ii) DDQ, dioxane, reflux; iii) isop
Lopenyl acetate, p-TsOH, BuOAc, reflux
(Recycle 3 times); iv) Ca (BH_Four)₂, MeOH
-EtOH, 0 → 4 ° C; v) PTAD, CH ₂Cl₂，
Room temperature; vi) a: TBSCl, imidazole, DMF, room
Warm, b: MeOH-THF containing 15% KOH, room temperature; vi
i) m-CPBA, CHCl₃, Room temperature; viii) a: TBA
F, THF, room temperature, b: 1,1,3,3-tetramethyl
Guanidine, 170 ° C (bath temperature); ix) NaBH_Four, J
Glyme, 80 ° C (bath temperature); x) a: TBSCl, a
Midazole, DMF, room temperature, b: Ac₂O, pyridine,
Room temperature; xi) a: hν, Et₂O, 0 ° C, b: hexane-
THF, room temperature; xii) a: TBSCl, imidazole, D
MF, room temperature, b: MeOH containing 5% KOH, 0 ° C .; xii
i) a: glutaric anhydride, pyridine, room temperature, b: TBA
F, THF, room temperature.

(2) Preparation of immunogen According to the flow chart shown below, the hapten derivative 1
A complex of the above hapten derivative and BSA was prepared using 4.1 μmol and 23.4 mg of BSA. The obtained complex had 17 molecules of hapten derivative bound to 1 molecule of BSA.

[0025]

[Chemical 3]

(3) Preparation of Antibody Rabbits were immunized with an emulsion of the above immunogen and complete Freund's adjuvant, and the resulting antiserum was treated with protein A.
By loading on a column, four types of antibodies (IgG fraction) were obtained.

Example 2: Properties of antibody The properties of the obtained antibody were evaluated by the RIA method shown in FIG. As a result, all the obtained antibodies had high titers (optimal dilution ratio: 1300 to 220,000 times), and the detection limit obtained from the dose response curve (Fig. 2) was 2.
10 pg / tube. In addition, known methods (A
nn. N. Y. Acad. Sci. , 51,660 (1
949)) calculated antibody affinity constant (Ka)
Was 0.34 to 3.3 × 10 ¹⁰ (M ⁻¹ ).

Furthermore, the cross-reactivity of antibodies can be determined by a known method (J. Clin. Endocrinol. Meta.
b. , 29,866 (1969)), as shown in the table below, 1α, 25 (OH) ₂ vitamin D ₃
Vitamin D ₃ with 100% reactivity to
The reactivity with the derivative was 6% or less.

[0029]

[Table 1]

Example 3 A kit comprising the following reagents for carrying out the RIA method shown in FIG. 1 was prepared. (RIA kits) antibody reagent (antibody of the present _{invention) 1α, 25 (OH) 2} D 3 standard solution tracer ([26,27- methyl - ³ H] -1α,
25 (OH) ₂ D _{3 If} necessary, dextran-
A charcoal solution or a second antibody solution may be attached.

[Brief description of drawings]

FIG. 1 illustrates a measurement method of RIA method.

FIG. 2 shows a dose response curve of the antibody of the present invention.

Claims (3)

[Claims] 1. A 1α, 25 (OH) having the following characteristics:
₂ Antibodies against vitamin D ₃ . Affinity constant (Ka value) 0.34 to 3.3 × 10 ¹⁰ (M ⁻¹ ) Cross reactivity 1α, 25 (OH) ₂ Vitamin D ₃ reactivity to 1
The reactivity to other vitamin D ₃ derivatives when it is set to 00% is 6% or less. _{2. A} method for measuring 1α, 25 (OH) ₂ vitamin D ₃ , which comprises using the antibody according to claim 1. 3. A kit for use in the measurement of 1α, 25 (OH) ₂ vitamin D ₃ , which comprises the antibody according to claim 1 as a constituent reagent.