JPH0717877A - Radioimmunoassay of 2'-5'oligoadenylate - Google Patents

Abstract

PURPOSE:To provide a detection method capable, e.g. of detecting disease by determining the oligoadenylic acid concentration in blood by radioimmunoassay with the use of a new oligoadenylic acid compound as an antigen for labeling with a radioactive substance. CONSTITUTION:In this method, 2'-5'oligoadenylate is determined by radioimmunoassay with the use of 5'-triphospho(adenyl 2'-5')2adenosine-beta- alanyltyrosine methyl ester of formula I as a compound for radioactive-<125>I- labeling. The compound of formula I is preferably obtained by the following process : oxidizing 5'-monophospho(adenyl 2'-5')2adenosine of formula II, e.g. by periodate oxidation, reacting the reactional product with beta-alanyltyrosine methyl ester in the presence of NaOH in DMF, reacting the produced 5'- monophospho(adenyl 2'-5')2adenosine-beta-alanyltyrosine methyl ester with N,N'- carbonylimidazole in a solvent in the presence of triethylamine and tri-n- octylamine and subsequently reacting the reactional product with pyrophosphoric acid.

Description

Detailed Description of the Invention

[0001]

The present invention relates to the novel 2'-5 'oligoadenylate compound production method thereof, and the compound ¹²⁵
The present invention relates to a radioimmunochemical assay method (radioimmunoassay method, hereinafter abbreviated as RIA) of 2′-5 ′ oligoadenylic acid (hereinafter abbreviated as 2-5A) used as an I-labeling antigen.

[0002]

2. Description of the Related Art An RIA utilizing the fact that an antigen labeled with a radioactive substance and an unlabeled antigen in a biological sample compete with each other for a specific antibody against the antigen, is a trace substance which is very excellent in sensitivity and specificity. It is widely used as a measurement method of.

RIA requires the use of radioactive materials,
¹²⁵ I is generally used as a radioactive substance for this purpose. ^For antigens that are difficult to label with ¹²⁵ I, ³ H,
Radioactive substances such as ¹⁴ C and ³² P are used for labeling. However, RIA using ³ H, ¹⁴ C, ³² P, etc.
Must use a liquid scintillation counter, with radioactive waste accompanied by organic solvents. In addition, ³ H, ¹⁴
Since C has a long half-life of radioactivity, it is difficult to dispose of radioactive materials. On the other hand, ³² P has a short half-life of radioactivity, which causes a problem in storage and handling.

In contrast, when labeling with ¹²⁵ I is possible with a well-type scintillation counter, it is not necessary to use an organic solvent as a scintillator, and since the half-life of radioactivity is relatively short, radioactive wastes Easy to process.

Labeling an antigen with ¹²⁵ I is relatively easy when it has a reactive group capable of introducing an iodine atom such as peptide, but when the antigen does not have a reactive group capable of introducing an iodine atom, As a labeling antigen, it is necessary to prepare a derivative in which a reactive group capable of being iodinated is introduced into an antigen site that does not inhibit binding to an antibody.

2-5A is a substance produced by ATP in the presence of double-stranded RNA by the 2-5A synthase introduced in vivo by the antiviral action of interferon. Therefore, measuring the concentration of 2-5A in blood is expected to be useful in knowing the function of the interferon-2-5A synthase system and also in diagnosing infectious diseases, and is of interest.

By the way, as a 2-5A RIA or radiobinding assay, a method using ³² P for labeling an antigen is described in Nature, 288 (13), 18
9-192, 1982; The Journal of Biological Ch
emistry, 259 (3), 1727-1730, 19
84 etc.

[0008]

However, these methods cannot be used as a method for measuring 2-5A in blood because they have a low specific emission of ³² P and cannot be used as a method for measuring 2-5A in blood, and no proposals have been made yet. Not not. Moreover, as described above, since ³² P is used, it is necessary to measure with a liquid scintillation counter, and the fact is that it is not suitable for application to clinical tests such as disease search. Further, since the method for labeling the ¹²⁵ I to 2-5A themselves are not known at present, yet it is difficult to synthesize ¹²⁵ 2-5A derivatives for I-labeled, it was used ^{1 25} I signs RIA has not been developed.

[0009]

[Means for Solving the Problems] In view of the above-mentioned circumstances, the present inventors have earnestly studied in order to find out a 2-5A derivative which can be labeled with ¹²⁵ I, and as a result, the radioactive substance in RIA (
We succeeded in synthesizing a novel 2-5A compound that can be used for labeling with ¹²⁵ I), and completed the present invention.

The 2-5A compound used for labeling in the present invention has a chemical structural formula (I)

[Chemical 2] 5'-triphospho (adenylyl 2'-5 ') represented by
₂ Adenosine-β-alanyl tyrosine methyl ester.

The novel 2-5A compound (I) is (a) the chemical structural formula (II)

[Chemical 3] The known compound 5′-monophospho (adenylyl 2′-5 ′) ₂ adenosine represented by is oxidized by, for example, a periodate oxidation reaction, and then in a dimethylformamide solution in the presence of sodium hydroxide. Reacted with β-alanyl tyrosine methyl ester to give chemical structure (III)

[Chemical 4] 5'-monophospho (adenylyl 2'-
5 ′) ₂ adenosine-β-alanyl tyrosine methyl ester step, (b) Compound (III) obtained in step (a) is dissolved in a solvent such as dimethylformamide, and triethylamine and tri-n-octylamine are dissolved. In the presence of N, N'-carbonyldiimidazole, and then with pyrophosphoric acid.

[0012]

EXAMPLES The novel 2-5A of the present invention will be described below based on examples.
Specific method for producing compound, analysis result of obtained compound, and ¹²⁵ I-labeling compound (antigen) for measuring 2-5A in blood by radioimmunoassay for the obtained compound
The usage method and the like used as will be described in detail.

Example 1 45 mg of a known compound 5'-monophospho (adenylyl 2'-5 ') ₂ adenosine represented by the chemical structural formula (II)
0.3 ml of an aqueous solution of (30 μmol) was mixed with 0.45 ml of a 0.1 M sodium periodate solution, and the mixture was stirred in an ice bath at 0 ° C. for 25 minutes. The reaction mixture was added to a solution prepared by dissolving 33 μl of 1M sodium hydroxide in 0.4 ml of a mixture of dimethylformamide: water (1: 1) and dissolving 30 μmol of β-alanyltyrosine methyl ester hydrobromide in 1M water. The mixture was stirred for 15 minutes in a 0 ° C. ice bath while maintaining the pH at 8.5 with sodium oxide. Then 0.
Add 0.6 ml of 5M sodium cyanoborohydride,
The mixture was stirred in an ice bath at 0 ° C for 1 hour while maintaining the pH at 6.5 with 1M hydrochloric acid. After confirming the formation of the reaction product by high performance liquid chromatography using RPC-5 column (HPLC), it QAE- Sephadex A-25 (HCO ₃ ^-
Type) column (30 cm x 15 mm inner diameter), 0.2
Elution with a 5 / 0.5 M linear concentration gradient of bicarbonate / triethylammonium (800 ml / 800 ml) buffer revealed that 5′-monophospho (adenylyl 2′-5 ′) ₂ adenosine corresponding to the chemical structural formula (III) was obtained. 39 mg (yield 75%) of triethylammonium salt of -β-alanyltyrosine methyl ester was obtained.

[0014]

[Chemical 5]

5'-corresponding to the above chemical structural formula (III)
Monophospho (adenylyl 2'-5 ') ₂ adenosine-β
-38 mg (24 μmol) of triethylammonium salt of alanyltyrosine methyl ester was added to 0.5 m of pyridine.
It is dissolved in 1 and water is distilled off azeotropically under reduced pressure. This operation was repeated 3 times, and then 0.5 ml of dimethylformamide
Was dissolved in 2 ml of dimethylformamide, and the residue was distilled off under reduced pressure. After adding 50 μl of triethylamine and 30 μl of tri-n-octylamine to this solution,
N, N'-carbonyldiimidazole 70 mg (0.4
mmol) was added and the mixture was stirred at room temperature for 2 hours. Next, 50 μl of dry methanol was added, and the mixture was stirred again at room temperature for 30 minutes.
To this, tributylammonium pyrophosphate 0.4 mm
was dissolved in 0.8 ml of dry dimethylformamide, and the mixture was stirred overnight at room temperature. The reaction solution was gradually added dropwise to a mixed solution of 5 ml of ethanol, 5 ml of acetone and 0.5 ml of acetone saturated with sodium perchlorate to form a white precipitate. The white precipitate was washed with acetone and ether and then vacuum dried. 25m of this dried product
It dissolves in 1 l, and QAE-Sephadex A-25 (HCO
₃ ⁻ type) column (30 cm × 15 mm inner diameter), 0.
Elution with a linear gradient of 33-0.67 M bicarbonate / triethylammonium (500 ml / 500 ml) gave the white product 5'-triphospho (adenylyl 2'-5 ') corresponding to formula (I). ₂ Adenosine-β-
Alanyl tyrosine methyl ester 13.8 mg (yield 3
0%).

[0016]

[Chemical 6]

Example 2 (1) Preparation of 2-5A antiserum 5'-triphospho (adenylyl 2'-5 ') ₃ adenosine 31 mg (15 μmol) was dissolved in 0.4 ml of water,
0.4 ml of 0.1M sodium periodate was gradually added while stirring in an ice bath at 0 ° C., and the mixture was stirred for 30 minutes. Then, a solution prepared by dissolving 20 mg of bovine serum albumin (hereinafter abbreviated as BSA) in 0.4 ml of a 0.2 M carbonate buffer (pH 9.2) was added, and the mixture was again stirred in an ice bath at 0 ° C. for 1 hour. Thereafter, 0.55 sodium cyanoborohydride (0.65 ml) was added, and a 1 M phosphate buffer solution (pH 5.
5) was added and the mixture was stirred for 30 minutes in an ice bath while maintaining the pH at 6.5, and then stirred overnight at 4 ° C. The reaction product was applied to a Sephadex G50 column (30 cm x 22 mm inner diameter) and eluted with 0.01 M phosphate buffer (pH 7.5), and the protein elution portion was collected to obtain a 1 mg / ml solution for immunization. It was Hara. The rabbit is immunized with this according to the usual method, and 2-5
A anti-rabbit serum stock solution was obtained. 0.25% of this antiserum stock solution
Diluted 10,000 times with 0.05M phosphate buffer (pH 8.0) containing BSA and 1% normal rabbit serum to give 2-5A.
It was used as an antiserum solution. (2) ¹²⁵ I-labeled 5'-triphospho (adenylyl 2 '
Preparation of -5 ′) ₂ adenosine-β-alanyl tyrosine methyl ester 5′-triphospho (adenylyl 2 ′ obtained in Example 1
-5 ′) ₂ adenosine-β-alanyl tyrosine methyl ester (I) in 0.05M phosphate buffer (pH 7.5)
50 μg / ml with 50 μg / ml, and 10 μl of this was labeled with ¹²⁵ I by the chloramine T method by a conventional method, and then purified with a Sephadex G10 column to obtain 1,200 Ci / mmo.
A ¹²⁵ I-labeled compound with a specific activity of 1 was obtained. 0.25
0.05M phosphate buffer (pH 8.0) containing% BSA
Was diluted to 30,000 cpm / 0.2 ml to prepare a ¹²⁵ I-labeled 2-5A solution ( ¹²⁵ I-labeled antigen).

Example 3 Method for measuring 2-5A in blood 2 mg of human blood was mixed with 2 mg of disodium ethylenediaminetetraacetate and immediately mixed by cooling and centrifugation at 4 ° C., and 100 μl of the obtained plasma sample was placed in a test tube. (2)
100 µl of the ¹²⁵ I-labeled 2-5A solution obtained in (1) and 200 µl of the 2-5A antiserum obtained in (1) of Example 2 were added, and the mixture was reacted at room temperature overnight. 0.1 ml of anti-rabbit γ-globulin goat serum was added to the obtained reaction solution, and the mixture was allowed to stand overnight and then centrifuged at 3,000 rpm to remove the supernatant by suction, and the amount of radioactivity in the precipitated portion was measured. At the same time, normal human plasma was subjected to charcoal treatment, and the obtained 2-5A free plasma was treated with 4 mg / ml of disodium ethylenediaminetetraacetate.
2-5A was added at a rate of 0, 0.0625, 0.
100 μ of each of 2-5A standard plasma (standard solution) added so as to have a concentration of 25, 1.0, 4 and 16 ng / ml.
1 was also measured by the same operation as above, and the standard curve shown in FIG. 1 was obtained. Thus, the concentration of 2-5A in the plasma sample was determined.

Example 4 Measurement of 2-5A in Blood of Patients with Various Diseases 13 bloods of patients with various diseases shown in Table 1 below.
As a control, the 2-5A concentration of each of 15 cases of normal human blood was measured by the method described in Example 3. The results are shown in Figure 2. 2-5A in blood in normal and patients
It can be seen that there is a difference in concentration.

[0020]

[Table 1]

[0021]

INDUSTRIAL APPLICABILITY As described above, an example in which the novel compound of the present invention is used as a ¹²⁵ I-labeling compound (antigen) for 2-5A measurement by RIA has been described.
It has been proved to be useful for enabling the measurement of -5A concentration and being suitable for application to clinical tests such as disease search.

[0022]

[Brief description of drawings]

FIG. 1 is a graph showing a standard curve for blood 2-5A measurement in Example 3, wherein the vertical axis represents ¹²⁵ I-labeled antigen binding rate (%),
The horizontal axis shows the 2-5A concentration (ng / ml).

FIG. 2 shows the results of measuring the 2-5A concentrations of blood samples of patients with various diseases and normal blood samples of Example 4 as subjects.

Claims (1)

[Reference number] P285 [Claims] 1. A chemical structural formula (I): 5'-triphospho (adenylyl 2'-
5 ′) ₂ adenosine-β-alanyl tyrosine methyl ester as a radioimmunochemical method for measuring 2′-5 ′ oligoadenylic acid using a radioactive substance-labeling compound (antigen)