JP2587239B2 - Enzyme-labeled thromboxane B (2) and method for measuring 11-dehydro-thromboxane B (2) using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to 11-dehydro-thromboxane B ₂ (hereinafter referred to as 11-dehydro-TXB ₂ ) by enzyme immunoassay (hereinafter abbreviated as EIA method). abbreviated.) used in quantifying, TXB ₂ class labeled with an enzyme, and assays related to 11-dehydro -TXB ₂ using the TXB ₂ class.

[Prior art] Thromboxane A ₂ (hereinafter abbreviated as TXA ₂ ) is one of the metabolites of the arachidonic acid cascade and is a compound having a strong platelet aggregation ability and a contractile action of vascular smooth muscle. Acad. Sci. USA, 72 , 2994 (1975)].

In recent years, the relationship between TXA _{2 in} a living body and various diseases has been discussed, and it has become very important to establish a method for accurately and easily measuring the concentration of TXA _{2 in} a living body.

However, TXA ₂ is a very unstable substance (eg, a half-life in a buffer solution near neutrality at 37 ° C. for 30 seconds), and is rapidly hydrolyzed to a stable inactive substance TXB ₂
Is converted to Furthermore the TXB ₂ are oxidized, it is metabolized to 11-dehydro -TXB _2.

Therefore, stable metabolites such as TXB ₂ and 11-dehydro-TXB ₂
It is conceivable that the concentration of TXA ₂ is measured indirectly by quantifying the concentration. Quantification of TXB _{2 by} the EIA method has been performed conventionally, but the measurement location is largely inaccurate and inaccurate. The reason for this is believed to be due to TXB ₂ to 11 dehydro -TXB ₂ unseparated in the specimen whether the cross-react with antibodies to TXB _2, or newly generated during blood collection is also measured together [ Abstracts of Kyoto Conference on Prostagland
ins, 7 (1984)].

Measuring the 11-dehydro -TXB ₂ by radioimmunoassay are already known but [Prostaglandins, 3
1 (5), 929 (1986)], has not been reported at all until now for the EIA method, it is therefore also TXB ₂ such that the enzyme-labeled used EIA method has not yet been known at all.

[Object of the invention]

The present inventors have found that 11-specific and selective EIA method dehydro -TXB ₂ among continue to develop, to find an optimum as the enzyme-labeled antigen 11-dehydro -TXB _2, in particular the cross-linking agent As a result of intensive studies, they found that the cross-linking condensation method using γ-aminobutyric acid was most excellent in terms of specificity and selectivity, and completed the present invention.Generally, haptens are labeled with enzymes. As a method,
Various methods are known, such as a direct bonding method and a bonding method with an appropriate cross-linking agent interposed therebetween. Although many are also known as crosslinking agents, there have been no reports on the use of γ-aminobutyric acid as a crosslinking agent. Furthermore, it was completely unpredictable to obtain an ideal enzyme-labeled antigen having an appropriate affinity for an antibody and excellent selectivity for a test substance.

[Configuration of the invention]

That is, the present invention relates to the general formula used in the EIA method. (In the formula, And R ¹ represents an enzyme in which one hydrogen atom of the amino group is removed and bonded. ] The enzyme-labeled TXBs ₂ represented by the formula:

In the general formula (I), Is preferred in each case. Especially But Enzyme labels representing the will, be preferred by measuring affinity is suitably of 11-dehydro -TXB ₂ to the antibody.

In the general formula (I), the enzyme represented by R ¹ is EI
Any enzyme commonly used in Method A may be used. Enzymes represented by R ¹ used in the present invention, for example, malate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, peroxidases,
Examples include acetylcholinesterase, alkaline phosphatase, glucoamylase, lysozyme, and β-D-galactosidase, with β-D-galactosidase being preferred.

(Production method of the present compound)

TXB ₂ represented by the general formula (I) can be produced by the following method.

[Wherein [A] and R ¹ represent the same meaning as described above. The compound represented by the general formula (II) is dissolved in a suitable organic solvent (preferably tetrahydrofuran) by dissolving the compound represented by the general formula (III) with a condensing agent such as trinormal butylamine and isobutyl chloroformate. It is obtained by reacting and further reacting with γ-aminobutyric acid. The reaction is performed, for example, at 4 ° C. for 1 hour. After the reaction, the product can be purified by a usual post-treatment.

The obtained compound represented by the general formula (II) is dissolved in a suitable organic solvent (preferably tetrahydrofuran) and reacted with a condensing agent such as tri-n-butylamine and isobutylchloroformate. The reaction is performed, for example, at 4 ° C. for 1 hour. Further, by reacting with β-D-galactosidase, the desired compound represented by the general formula (I)
Enzyme-labeled TXB ₂ compound represented in is obtained. After the reaction, the product is fractionated by gel chromatography to recover the TXB ₂ compounds represented by the general formula (I) having enzymatic activity.

The enzyme-labeled TXBs ₂ represented by the general formula (I) of the present invention are EI
It can be used as an enzyme-labeled antigen in the case of quantifying the 11-dehydro -TXB ₂ in Method A. The first antibody solid phase method as the EIA method, the second Antibodies solid phase method and liquid phase method are known, in TXB ₂ class is one of the methods of the present invention, specific and highly selective enzymatic label It can be used as an antigen.

In order to carry out the EIA method using the enzyme-labeled antigen represented by the general formula (I), an antiserum diluted to an optimum concentration, a sample or a standard, and the enzyme-labeled antigen represented by the general formula (I) are optimally used. After incubating at a temperature, the resulting antigen-antibody complex is separated into a free form and a bound form by a two-antibody method or the like, and then the enzyme-substrate is allowed to act on the antigen-antibody complex to obtain the target from the fluorescence intensity of the product. it is possible to determine the concentration of 11-dehydro -TXB _2. In carrying out the EIA method, the biological sample is subjected to pretreatment, if necessary, to be purified to such an extent that the antigen-antibody reaction is not inhibited, and then used for measurement.

The antiserum used in the EIA method can be obtained by a known method. That is, it can be obtained by emulsifying an antigen in an appropriate adjuvant, for example, complete Freund's adjuvant, and sensitizing a host animal, for example, rabbit, guinea pig or the like. Antigen 11-dehydro -TXB ₂ and protein as haptens, obtained by condensing for example bovine serum albumin (BSA) by a known method. The enzyme-labeled antigen used in the present invention is not limited to these,
When γ-aminobutyric acid is used as a cross-linking agent, a good antigen with high specificity can be obtained.

〔Example〕

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Enzyme labeling using γ-aminobutyric acid (GABA) as a crosslinking agent
TXB ₂ [in the general formula (I) But In which R ¹ represents β-D-galactosidase] and tetrahydrofuran (50 μl) containing TXB ₂ (100 μg).
), Trinormal butylamine (2.5 µ), isobutyl chloroformate (1.3 µ) and γ-aminobutyric acid (100 µg) were added, and the reaction solution was adjusted to 100 µ with tetrahydrofuran, followed by stirring at 4 ° C for 1 hour. . After removing unreacted GABA, tetrahydrofuran (50 μL) containing the obtained condensate (100 μg as TXB ₂ )
μ), trinormal butylamine (2.5 μ) and isobutyl chloroformate (1.3 μ) were added, and the reaction solution was adjusted to 100 μ with tetrahydrofuran and reacted at 4 ° C. for 1 hour with stirring. Next, 0.5 ml of a 0.5% aqueous sodium hydrogen carbonate solution containing 1 mg of β-D-galactosidase
The acid anhydride reaction solution obtained above was added dropwise to the mixture at a rate of 10 μm per minute while stirring, and reacted for 2 hours after the completion of the addition. After the reaction was completed, tetrahydrofuran was distilled off from the mixed solution, followed by gel filtration to collect a protein fraction. β-
The molecular weight of D- galactosidase, calculated as 540000, beta-D- galactosidase per mole TXB ₂ 7.4 mol was condensed.

Example 2 Enzyme labeling using γ-aminobutyric acid (GABA) as a crosslinking agent
11-dehydro-TXB ₂ [in the general formula (I) And a compound wherein R ¹ represents β-D-galactosidase.] A protein fraction was recovered in the same manner as in Example 1 using 11-dehydro-TXB ₂ (100 μg).

When calculating the molecular weight of beta-D-galactosidase as 540000, beta-D-galactosidase per mole 11- dehydro -TXB ₂ 7.2 mol was condensed.

Example 3: 11-dehydro -TXB ₂ antiserum 11- dehydro -TXB ₂ (24.74mg) 0.01M phosphate buffer containing (pH 5.5; 10 ml) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide; phosphoric acid containing (ECD 50.5 mg) A buffer solution (1 ml) was added, and then a buffer solution (3 ml) of the same composition containing bovine serum albumin (100.5 mg) was added.
For 24 hours. As the condensate, a protein fraction was recovered by a gel filtration method.

For sensitization to rabbits, 1 mg of the hapten antigen obtained above was administered as a BSA amount per sensitization. That is, 1 mg of the hapten antigen obtained above was dissolved in 0.5 ml of physiological saline, and an emulsion was prepared with Comple- Freund's adjuvant (1 mg). This emulsion (1.5 ml) was intradermally administered to rabbits. The sensitization interval was performed every two weeks. An increase in the antibody titer was observed in a test blood sample 13 days after the fourth sensitization.

Example: 11-dehydro-TXB ₂ using various enzyme-labeled antigens
Using the EIA (double antibody method) antiserum prepared in Example 1 with the enzyme labeled antigen of the invention and prepared in Example 2 3 actually 11-dehydro -TXB ₂ measurements EIA (double antibody Method).

This experiment was performed on four types of enzyme-labeled TXBs ₂ (Control 1 to Control 4) prepared by the following method in addition to the enzyme-labeled TXBs ₂ of the present invention.
- to compare the sensitivity of dehydro -TXB _2.

Control 1: Enzyme-labeled 11-dehydro-TXB ₂ without cross-linking agent 1 ml of 0.1M phosphate buffer (pH 6.8) containing 11-dehydro-TXB ₂ (100 μg) and the same buffer containing ECD (1 ml) (1 ml) 0.5m
l) was added and stirred at room temperature for 2 hours. Β-D
-A phosphate buffer (0.5 ml) of the same composition containing galactosidase (1 mg) was added, and the mixture was stirred at 4 ° C for 24 hours. It was obtained following enzyme-labeled 11-dehydro -TXB ₂ by gel filtration condensates.

Control 2: The following enzyme labels carried in the same manner as the Control 1 using an enzyme-labeled TXB ₂ TXB ₂ containing no crosslinking agent
TXB ₂ was obtained.

Control 3: Enzyme-labeled 11-dehydro-TXB ₂ using leucine as a cross-linking agent The following enzyme-labeled 11-dehydro-TXB ₂ was obtained in the same manner as in Example 1 using leucine as a cross-linking agent.

Control 4: glycine with an enzyme-labeled 11-dehydro -TXB ₂ glycine to crosslinking agent as a crosslinking agent, to obtain the following enzyme-labeled 11-dehydro -TXB ₂ in the same manner as in Example 1.

Experimental method and results: Phosphate buffer (100μ) containing sample or standard,
After adding the diluted antibody (100 μ) and the phosphate buffer (100 μ) containing the enzyme label shown in Example 3, and mixing,
Incubated for 1 hour at 7 ° C. Next, diluted normal rabbit serum (100 μ) and anti-rabbit IgG (100 μ) were added, and 4
Left overnight at ° C. After the completion of the reaction, the reaction solution was 3,000 rp at 4 ° C.
After centrifugation for 10 minutes and removing the supernatant by suction, 0.1 mM 4
-Methyl-umbelliferyl β-D-galactoside (0.
2 ml) and reacted at 37 ° C. for 30 minutes. After adding 0.1 M glycine buffer (3.0 ml) to stop the reaction,
Centrifuge at 0 rpm for 10 minutes and measure the fluorescence intensity of the supernatant at the excitation wavelength λEx
The measurement was performed at 330 nm and the fluorescence wavelength λEm460 nm. Standard 11-dehydro
A calibration curve prepared based on measurements for -TXB ₂ shown in Figure 1.

Figure 1 is a calibration curve showing the relationship between concentration and binding rate of 11-dehydro -TXB ₂ in the sample. A calibration curve (shown by -O-) measured using the enzyme label prepared in Example 1 using TXB ₂ as a hapten, and 11-dehydro-TXB ₂
The calibration curves (indicated by-●-) measured using the enzyme-labeled product prepared in Example 2 using as a hapten showed a good linear relationship, indicating that 11-dehydro- It can be seen that TXB ₂ can be accurately measured down to low concentrations. Among the six enzyme labels, the enzyme label using γ-aminobutyric acid prepared in Examples 1 and 2 as a cross-linking agent showed remarkable improvements in sensitivity and calibration range, and was useful for the present invention. Sex was proved.

[Brief description of the drawings]

FIG. 1 is a graph showing a calibration curve in EIA when six types of enzyme labels are used.

Claims (5)

(57) [Claims] (1) General formula (In the formula, And R ¹ represents an enzyme in which one hydrogen atom of the amino group is removed and bonded. Enzyme-labeled thromboxane B ₂ compounds represented by]. 2. In the general formula (I) Enzyme-labeled thromboxane B ₂ such range first claim of claims is. 3. In the general formula (I) Enzyme-labeled thromboxane B ₂ such range first claim of claims is. 4. The method according to claim 1, wherein R ¹ in the formula (I) is a residue of β-D-galactosidase.
The enzyme-labeled thromboxane B according to any one of the above items
Class ₂ . 5. A compound of the formula (I) (In the formula, And R ¹ represents an enzyme in which one hydrogen atom of the amino group is removed and bonded. ] Enzyme-labeled thromboxane B ₂ compounds represented by and 11-dehydro - thromboxane hexane B ₂ by double antibody method using the antibody of the hapten 11-dehydro - method of measuring thromboxane B _2.