JP2517561B2 - Category Estrogen Immunoassay Kit - Google Patents

Description

TECHNICAL FIELD The present invention relates to a catechol estrogen immunoassay. More specifically, it relates to a catechol estrogen immunoassay kid.

(Background Art) Catechol estrogen is one of the major metabolites of estrogen, which is an important hormone related to breast cancer, and is a substance that is drawing attention from the viewpoint of physiology and metabolism.

Regarding the similarity with estrogen, it has already been reported that catechol estrogen may act in a promoting manner on breast cancer cells and may act in a suppressive manner. It is known to be a substance that is deeply involved in.

On the other hand, regarding this catechol estrogen, there is a growing interest in its specific actions in the central and peripheral areas such as physiological actions in the brain such as the influence on luteinizing hormone and prolactin secretion, and uterotropic action.

However, this catechol estrogen is present in the body in a very small amount (of the order of femtomoles), and therefore its detection is extremely difficult, and it has been difficult to accurately determine the physiological and metabolic actions of catechol estrogen.

Therefore, it has been strongly desired to realize a method for detecting a trace amount of catechol estrogen.

This is especially important for future diagnosis of breast cancer. If the relationship between catechol estrogens and breast cancer can be accurately determined, it is now clear that the hormonal control by ovarian ablation and its application to the treatment of breast cancer are not always clearly clarified. Become. With proper diagnosis of breast cancer,
It will bring a great gospel to the decision of ovariectomy from breast cancer patients.

(Object of the Invention) The present invention has been made in view of the above circumstances, and is an immunoassay for catechol estrogen, which enables detection and measurement of an extremely small amount of catechol estrogen, which is attracting attention for its physiological and metabolic effects. Intended to serve Kid.

DISCLOSURE OF THE INVENTION The immunoassay kid of the present invention is for the production of antibodies having specificity for catechol estrogen,
The greatest feature is that the protein complex of catechol estrogen represented by the following formula is used as one element.

(A in the formula is = N-O- or -O-CO-, and n is 1
To 4 integer, -NH-P is the residue obtained by removing one hydrogen atom of the amino group from a protein, R ₁ and R ₂ are each different, represent H or OH in) protein complex of this catechol estrogens ( i) is, for example, A = N—O—, n is 1, —NH—
When P is a bovine serum albumin residue (ii), 2
It can be produced from -hydroxyestrone or 4-hydroxyestrone (iii) according to the following reaction formula.

That is, 2-hydroxyestrone or 4-hydroxyestrone (iii) is condensed with carboxymethylhydroxylamine to give 2-hydroxyestrone-17- (O-carboxymethyl) oxime or 4-hydroxyestrone-17- (O-carboxymethyl) oxime
Hydroxyestrone-17- (O-carboxymethyl)
It is induced by oxime (iv) and bound to bovine serum albumin (BSA) as a carrier protein to synthesize the target protein complex hapten-BSA (ii). The starting material 2-hydroxyestrone or 4-hydroxyestrone (iii) was synthesized from estrone (3-hydroxyestra-1,3,5 (10) -trien-17-one) (v) according to the following reaction formula. can do.

The catechol estrogens to be the targets of the immunoassay of the present invention are four substances (catechol estradiol and catechol estrone) of (vi) and (vii) in the formula.

The protein that forms the —NH—P group represented by the formula (i) of catechol estrogen used for antibody production of the present invention is not limited to bovine serum albumin, and is commonly used for haptens in the field of immunochemistry. There is no particular limitation as long as it is provided. Usually, albumin, globulin, etc. are used. Particularly, bovine serum albumin and rabbit serum albumin are preferably used.

To produce an antibody specific to catechol estrogen using the obtained catechol estrogen-protein complex (i), the complex (i) is treated with an animal (cattle, horse, sheep, goat, rabbit, It is parenterally administered to a living body of a rat, a mouse, etc., and an antibody is produced in the living body. Then, body fluid (for example, blood) is collected from the living body. Usually, it is used in the form of serum containing antibody, that is, antiserum.

In the present invention, the catechol estrogen immunoassay is carried out using the above-mentioned antibody. As the labeled antigen used in combination with the antibody, catechol estrogen labeled by an appropriate method is used. For this labeling, radioactive substances, enzymes, fluorescent substances, luminescent substances,
An appropriate one such as can be used. In addition, the position of introduction of these labeling substances into catechol estrogen is not particularly limited, as long as the immunoactivity of the obtained labeled antigen is retained. Labeling with radioactive elements such as ³ H, ¹⁴ C, ¹³⁵ I and ¹³¹ I is also effective.

The immunoassay kid of this invention consists of these labeled catechol estrogens and the antibodies described above. This immunoassay kid is particularly useful for detecting and quantifying catechol estrogen properties that are presumed to be involved in the development, proliferation, or suppression of breast cancer. In addition, it is not limited to breast cancer, and is also essential for determining the physiology and metabolism of various tissues including the brain of the living body.

Regarding breast cancer, the occurrence of cancer, its progression,
Furthermore, it is suggested that it gives an important index for the judgment of ovariectomy. In this regard, the present invention is also useful as a method for assaying the enzymatic activity of catechol estrogen sulfotransferase and catechol estrogen sulfatase, or an assay kid, as in the case of estrogen, which has been suggested to be associated with breast cancer.

Hereinafter, the present invention will be described in more detail with reference to examples. The present invention is not limited to these examples. Specific operating conditions of the immunoassay can be appropriately selected.

Example 1 (Synthesis of catechol estrogen antibody-producing reagent) (a) 100 mg of 2,3-dihydroxyestra-1,3,5 (10) -trien-17-one (formula (iii)) and 50 mg of ascorbic acid were added to 10 ml of methanol. , 100 mg of carboxymethylhydroxylamine hydrochloride was added, and the mixture was stirred overnight at room temperature in a nitrogen stream.

The solvent of the reaction solution was distilled off under reduced pressure, and 0.05N-hydrochloric acid 1 was added to the residue.
0 ml was added, and the mixture was extracted 3 times with ethyl acetate.
The organic layer was washed twice with water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure.

The residue was recrystallized from acetic acid ethyl ester-ether,
60 mg of 2-hydroxyestrone-17- (O-carboxymethyl) oxime (formula (iv)) was obtained as yellow needles.

(B) 50 mg of the above product oxime (formula (iv)) is dissolved in 2 ml of dioxane, 70 mg of bovine serum albumin (BSA) and 100 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride 1M phosphate buffer solution. The mixture was added dropwise to a 40 ml (pH 6.5) solution, protected from light, and stirred overnight at pH 6.5 and 40 ° C.

The reaction solution was dialyzed in 0.001% ascorbic acid 2 for 1 hour, and the dialysis operation was repeated, and after freeze-drying, 120 mg of hapten-BSA (formula (ii), R ₁ = OH, R ₂ = H) was obtained as a pale yellow powder. Obtained.

Example 2 (Production of anti-catechol estrogen antiserum) 4 mg of the hapten-BSA obtained in Example 1 was dissolved in 0.5 ml of physiological saline for injection, and 0.5 ml of Freund's Complete adjuva-nt was added. 30
Stir vigorously for a minute.

1 ml of the suspension was subcutaneously administered to white male rabbits (1.5 to 2.5 kg) every two weeks for two months, and once a month thereafter thereafter to the legs and back. Two months after the start of administration, 8 to 10 days after the last administration every month, blood is collected from the ear vein (1 to 2 m).
l) did.

After leaving it at room temperature for 1 hour, it was centrifuged at 800 xg and 4 ° C for 15 minutes to obtain an antiserum.

After 7 to 8 months, 8 days after the final administration, whole blood was collected from the carotid artery, and antiserum was obtained by the same procedure as above. The obtained antiserum was stored by cooling at -20 ° C.

Example 4 (Antibody titer and dilution of antiserum) The titer of the antiserum obtained in Example 3 was x50, x
A standard curve was prepared with 100, × 200, × 500, × 1000, and × 5000 dilutions.

 The antibody titer was measured as follows.

[ ³ H] catechol estrogen (approximately 5,000 dpm) and catechol estrogen (0.05 to 1.0 ng), diluted antiserum (1:50, 100, 200, 500, 1,000, 5,000) 0.05 M borate buffer (pH 7.4) ) (0.05% BSA, 0.06% bovine serum γ-globulin, 0.01% NaN ₃ , 0.05% ascorbic acid) solution (0.25
ml) was added and incubated at a temperature of 37 ° C. for 1 hour.
Then 50% (NH ₄ ) ₂ SO ₄ (0.25 ml) was added, stirred and left at room temperature for 15 minutes.

After centrifugation at 800 xg for 15 minutes, the supernatant (0.2m
A scintillator (10 ml) was added to l) and the radioactivity was measured with a liquid scintillation counter.

In this way, the antibody titer was determined as the dilution ratio showing 50% binding to the antiserum. At this time, the optimum dilution ratio for the assay was determined and a standard curve was prepared.

2-Hydroxyestrone had a difference of inhibition (%) of × 200, × 500, and × 2,000-fold diluted antiserum in the measurement range of 0.05 to 1.0 ng of 20, 40, 0 (%), respectively. A standard curve was prepared by setting the fold to the optimum dilution rate (Fig. 1 of the attached drawings). Also, of 4-hydroxyestrone
Optimal dilution ratio of antiserum in the measurement range of 0.05 to 1.0 ng is 500
It was twice (see FIG. 2).

Example 5 (Cross Reactivity / Radioimmunoassay) The obtained antiserum was examined for cross reactivity with other catechol estrogens.

That is, [6.7- ³ H] 2-hydroxy-estrone or [6.7- ³ H] when using the 2-hydroxy estradiol, 4 kinds of catechol estrogens to anti hydroxy estrone antiserum (2-hydroxy estrone, 2-hydroxy estradiol, Cross-reactivity of 4-hydroxyestrone, 4-hydroxyestradiol) was determined by radioimmunoassay.

 Radioimmunoassay was performed by the following method.

Dilute antiserum (1: 500) (0.25 ml) was added to [ ³ H] catechol estrogen (approximately 5,000 dpm) and catechol estrogen (0.05-10.0 ng) or a sample, and the mixture was incubated at 4 ° C for 18
Incubated for hours. Then 50% (NH ₄ ) ₂ SO ₄ (0.2
5 ml) was added, and the mixture was stirred and allowed to stand at room temperature for 15 minutes.

After centrifugation at 800 xg for 15 minutes, a scintillator (10 ml) was added to the supernatant (0.2 ml), and radioactivity was measured with a liquid scintillation counter.

[6.7- ³ H] 4-hydroxy-estrone or [6.7-
^{The same} was done for ³ H] 4-hydroxyestradiol.

Cross-reactivity is 50% of labeled antigen for antiserum binding
The cross-reactivity was calculated by the value indicating inhibition.

As shown in the following table, each showed almost 100% cross-reactivity.

This is an antiserum obtained by immunizing with the antigen of Example 1 condensed with BSA via the carbonyl group at the 17-position, and therefore changes in the carbonyl group or the hydroxyl group at the 17-position on the structure of estrogen are observed. It is not affected. Therefore, the assay is possible as the total amount of catechol estrogen composed of catechol estrogen and catechol estradiol.

Example 6 (Catechol Estrogen Sulfotransferase Assay) Using four catechol estrogens as substrates,
The amount of catechol estrogen sulfate conjugate produced by sulfotransferase in the cytoplasmic and cell membrane fractions was measured by radioimmunoassay to determine the activity of this enzyme. The method established by Examples 4-5 was used for the radioimmunoassay.

That is, catechol estrogen (0.5-10 μM)
And PAPS (40 μM), breast cancer cells (rat breast cancer)
Cytoplasmic fraction (0.5 mg / 0.5 ml) or cell membrane fraction (0.5
(mg / 0.5 ml) was added and incubated at 37 ° C for 30 minutes.
Then, extract with ether (0.5 ml x 2), and extract the water layer (0.05 m
Arylsulfatase (10 μg) 0.2 M acetate buffer (pH 5.5) solution (0.5 ml) was added to the above l), and the mixture was incubated at 55 ° C. for 1 hour. Further, the reaction solution was extracted with ether (0.5 ml × 2), and the organic layer was dried with nitrogen gas.

The amount of catechol estrogen was measured according to Example 5. At the same time, as a blank test, a test using each heat-treated fraction was also performed to correct the measured values.

For 4-hydroxyestrone, the amount of sulfate conjugate produced by this assay was as shown in FIG. The results of the addition recovery test are shown in the following table.

2-hydroxyestrone-mono-sulfate, 4
-Hydroxyestradiol-mono-sulfate is
In the range of 10 to 50 ng, 2-hydroxyestradiol-mono-sulfate, 4-hydroxyestrone-
It was revealed that mono-sulfate had a constant recovery rate in the range of 5 to 50 ng and could be quantified with good reproducibility.

In addition, when Vmax (maximum enzyme rate) and Km (Mikhail constant) of catechol estrogen sulfotransferase were determined, it was found that this enzyme was localized in the cytoplasmic fraction.

[Brief description of drawings]

1 and 2 show the standard curves of 2-hydroxyestrone and 4-hydroxyestrone, respectively. FIG. 3 shows the relationship between 4-hydroxyestrone and the amount of sulfate conjugate formation.

Claims (4)

(57) [Claims] 1. The following formula (A in the formula is = N-O- or -O-CO-, and n is 1
To 4 integer, -NH-P is the residue obtained by removing one hydrogen atom of the amino group from a protein, R ₁ and R _2, the antibody-producing reagent represented by each represents H or OH in different,) A catechol estrogen immunoassay kid comprising an antibody having specificity for catechol estrogen produced by using it as an antigen, and a labeled catechol estrogen. 2. The immunoassay kit for catechol estrogen according to claim 1, which uses catechol estrogen labeled with a radioisotope. 3. The immunoassay kid of catechol estrogen according to claim 1, which is used for detecting catechol estrogen properties in breast cancer. 4. A catechol estrogen immunoassay kid according to claim (1) or (2), which is used for diagnosis of breast cancer.