JPH0123060B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying adrenergic _β2 receptor-stimulating bronchodilators (β-stimulants) by enzyme immunoassay (hereinafter referred to as EIA).

Conventionally, various bronchodilators that stimulate adrenergic beta receptors have been used clinically, but recently developed clenbuterol, which is represented by the following formula (a) (Reference: Arznein.-Forsch., Vol . 26 , p. 1404) (1976)) and its analogues represented by the following formulas (b) and (c): It is a therapeutic agent for bronchial asthma and chronic bronchial asthma that has particularly excellent bronchodilatory effects.

That is, clenbuterol and its analogs have high selectivity for adrenergic β ₂ receptors and therefore act selectively on the bronchus, while their effects on the heart through stimulation of β ₁ receptors are low. and the clinical dose of such clenbuterone and its analogs is
The dose of clenbuterol in humans is significantly lower than that of conventional β-stimulants, for example, 20 to 40 μg per dose.

On the other hand, pharmacological action (or side effects) generally has a correlation with the blood concentration and tissue concentration of a drug, and in particular, measurement of the blood concentration of a drug in humans is clinically extremely meaningful. Because the clinical doses of clenbuterol and its analogs are extremely low, its concentration in body fluids cannot be measured using conventional measurement methods.

The present inventors have arrived at the present invention as a result of extensive research into ultratrace quantitative methods for measuring the concentration of clenbuterol and its analogs in body fluids.

That is, the present invention relates to an EIA method for clenbuterol and its analogues. According to the present invention, the drug concentration in a sample can be measured with extremely high sensitivity and good reproducibility, and the operation method is also simple. This allows for rapid processing of large numbers of samples.

In addition, radio immunoassay (Radio
It does not require special facility equipment such as the immunoassay method, and can be carried out using very common equipment and equipment.

According to the present invention, the following formula [] [In the formula, R ₁ and R ₂ represent a chlorine atom or a trifluoromethyl group, and X represents -N ₂ ⁺ . ] An enzyme-labeled antigen obtained by covalently bonding a clenbuterol represented by the following and an enzyme via the group x of the clenbuterol.

Antibiotics obtained by covalently bonding a clenbuterol represented by the above formula [] with a polymer compound via the group x of the clenbuterol and inducing a hapten antigen by parenterally administering it to an animal. Hapten antibody and at least the following reagent (a) An enzyme-labeled antigen obtained by covalently bonding a clenbuterol represented by the above formula [] and an enzyme via the group x of the clenbuterol.

(b) A hapten antigen obtained by covalently bonding a clenbuterol represented by the above formula [] to a polymer compound via the group x of the clenbuterol is parenterally administered to an animal to induce the antigen. A kit for an enzyme immunoassay of clenbuterol is provided, which is comprised of an anti-hapten antibody, (c) a carrier adsorbed with a second antibody.

According to the EIA method of the present invention, drug concentrations of β-adrenergic receptor-stimulating bronchodilators, particularly clenbuterol of the above formula (a) and its analogues of the above formulas (b) and (c), can be determined with extremely high sensitivity. It can be measured with good reproducibility.

Generally, the principle of EIA is to mix an enzyme-labeled antigen, its corresponding antibody, and a analyte (unlabeled antigen) to perform a competitive antigen-antibody reaction. B) and the free enzyme-labeled antigen that did not bind to the antibody (hereinafter abbreviated as F) are separated, the labeled enzyme activity in F or B is measured, and the labeled enzyme activity is determined from the calibration curve prepared in advance. This is done by quantifying the antigen in the specimen. In the EIA of the present invention, the analyte is an adrenergic beta receptor-stimulating bronchodilator such as clenbuterol or its analogues, and such a compound specifically binds to the corresponding antibody, but it is not allowed to inject itself into the animal. It does not induce the production of the corresponding antibody even when administered parenterally, and is therefore a so-called hapten. Enzyme-labeled antigens are enzyme-labeled antigens obtained by covalently bonding clenbuterols represented by the above formula [] with an enzyme, and antibodies are obtained by covalently bonding clenbuterols represented by the above formula [] with a polymer compound. Antibodies (so-called anti-hepten antibodies) can be obtained by parenterally administering the resulting conjugate (so-called hapten antigens) to animals to induce them. Further, in the present invention, separation of F and B is carried out by the so-called two-antibody method.

As mentioned above, the enzyme-labeled antigen of the present invention has the following formula [] [In the formula, R ₁ and R ₂ represent a chlorine atom or a trifluoromethyl group, and X represents -N ₂ ⁺ . ] It is obtained by covalently bonding a clenbuterol represented by the following and an enzyme via the group x of the clenbuterol. Further, anti-hapten antibodies can be induced by parenterally administering to animals a hapten antigen obtained by covalently bonding clenbuterol represented by the above formula [] with a polymer compound via the group x of the clenbuterol. This is obtained by

In the above formula [], R ₁ and R ₂ are a base atom or a trifluoromethyl group, and x is a diazonium salt (-N ₂ ⁺ ). The compound of the above formula [] is
For example, clenbuterol or the above formula [b],
By reacting the analogue of [c] with sodium nitrite in an acidic solution of hydrochloric acid by a conventional method, a compound in which x is -N ₂ ⁺ in the above formula [] can be obtained.

Specific examples of clenbuterols represented by the above formula [] include the following formula: Clenbuterols represented by:

In EIA, the measurement sensitivity, reproducibility, etc.
Whether the test drug and anti-hapten antibody have specific binding ability or whether the anti-hapten antibody is approximately equivalent to the enzyme-labeled antigen and unlabeled antigen, that is, the antigen in the test sample or the standard antigen for creating a calibration curve. It depends on whether or not it has a strong binding ability. Then, as in the present invention, by using clenbuterol of the above formula [] and preparing an enzyme-labeled antigen or an anti-hapten antibody as shown below, an enzyme-labeled antigen having a binding ability that satisfies the above requirements,
Anti-hapten antibodies are obtained.

Furthermore, in the present invention, clenbuterol or any of its analogs can be quantified by using enzyme-labeled antigens, anti-hapten antibodies, etc. prepared using any of the compounds represented by the above formula []. Is possible. The enzyme to be covalently bonded to clenbuterol when obtaining an enzyme-labeled antigen has high substrate specificity, allows easy measurement of its activity with high sensitivity, and is stable even after bonding with clenbuterol of the above formula []. In addition, it is necessary to select an enzyme that does not have a substrate for the enzyme or a substance that inhibits its enzyme activity in the test sample. Examples include phosphatase and peroxidase, and among these, β-D-galactosidase is particularly preferred. The clenbuterol of the above formula [] and the enzyme react with the phenol residue and histidine residue present in the enzyme molecule via the -N ₂ ⁺ of the group x of the above formula [] to form a covalent bond. , becomes an enzyme-labeled antigen.

As described above, the anti-hapten antibody used in the present invention is induced by parenterally administering to animals a hapten antigen obtained by covalently bonding clenbuterol of the above formula [] with a polymer compound. .

Here, preferred examples of the polymer compound include human serum albumin (HSA) and bovine serum albumin (BSA).

A hapten antigen can be obtained by covalently bonding such a polymer compound with clenbuterol of the above formula [] by a method substantially similar to the method for preparing the enzyme-labeled antigen described above. This hapten antigen is administered parenterally to an animal in a conventional manner with an adjuvant such as Freund'scomplete adjuvant, i.e., injected intradermally or subcutaneously into the animal to produce the corresponding anti-hapten antibody, and then blood is collected, followed by ammonium sulfate. Anti-hapten antibodies can be prepared by obtaining an IgG fraction by means such as fractionation or gel filtration. Or take blood again
Serum can also be obtained and used as is. Examples of animals used here include rabbits, goats, and guinea pigs, with rabbits being preferred. In addition, when collecting blood, it is recommended to periodically measure the blood antibody titer and collect all the blood at the point when the titer is highest.

In this way, an enzyme-labeled antigen and an anti-hapten antibody are obtained, and after a competitive antigen-antibody reaction is performed by mixing the enzyme-labeled antigen, anti-hapten antibody, and analyte (i.e., unlabeled antigen), the enzyme bound to the antibody is Enzyme-labeled antigen that did not bind to labeled antigen (B)
(F) and preferably B in F or B.
Quantification of EIA is performed by measuring the enzyme activity in the specimen and quantifying the antigen in the specimen from a standard curve prepared in advance.

In the present invention, F and B are separated by a two-antibody method. The second antibody used in the two-antibody method is obtained by parenterally administering the polymer compound used to prepare the anti-hapten antibody to an animal to induce the antibody, and then parenterally administering the antibody to the animal. Using elicited antibodies. Usually, the second antibody is preferably produced using a goat if the animal that produced the first antibody is a rabbit. Alternatively, an IgG fraction of anti-rabbit IgG induced by parenterally administering rabbit IgG to goats can also be used as the second antibody.

To separate F and B using this second antibody, the second antibody is physically adsorbed on a suitable carrier, and B is insolubilized by binding to the second antibody adsorbed on the carrier. The so-called two-antibody solid-phase method is suitable. Examples of the carrier used here include polystyrene balls, polystyrene plates, polystyrene tubes, glass plates, silicon pieces, and silicon tubes.

EIA is quantified by measuring the enzyme activity in B thus separated.

It is convenient to handle the enzyme-labeled antigens, anti-hapten antibodies, etc. mentioned above in the form of kits.

Therefore, according to the present invention, at least the following reagents (a) an enzyme-labeled antigen obtained by covalently bonding a clenbuterol represented by the above formula [] and an enzyme via the group x of the clenbuterol; (b) An anti-hapten obtained by parenterally administering to an animal a hapten antigen obtained by covalently bonding a clenbuterol represented by the above formula [] to a polymer compound via the group x of the clenbuterol to induce the antigen. antibody.

(c) A kit for enzyme immunoassay of clenbuterol and its analogs is provided, which comprises a carrier to which a second antibody is adsorbed.

The above reagents (a), (b), used in these kits
(c) is produced by the method described above.

A buffering agent, a standard solution of clenbuterol or its analogue, a reagent for enzyme quantification, etc. are added to such a kit. Examples of the enzyme quantitative reagents include substrates for enzyme activity measurement, coloring agents, enzyme reaction terminators, and the like.

As described above, the present invention provides a preferred EIA method for clenbuterol, which is an adrenergic β receptor-stimulating bronchodilator, or its analogs.

The present invention will be explained in more detail below with reference to Examples.

Reference example [Clenbuterol-HSA manufacturing method] Put 0.4 ml of pure water in a small flask, dissolve 3 mg of clenbuterol in it, and then add INHcl to adjust the pH of the solution to 1.5. Cold, under stirring (under bright light) NaNO ₂ solution 0.2 ml (NaNO ₂ 15 mg/ml H ₂ O)
dropwise and allow to react for about 30 minutes. Slowly add ammonium sulfamate solution dropwise to decompose excess nitrosonium ions.

The solution of the diazotized form of clenbuterol thus obtained was added to a phosphate buffer solution of HSA (0.1 molar solution,
PH7.4) Gradually drip to 1ml.

The reaction was stirred in the cold overnight while maintaining the pH at 7.5 with IN NaOH.

After the reaction is complete, add phosphate buffer (0.01 mol, PH7.5)
Clenbuterol-HSA solution is obtained by performing dialysis using as an external dialysis fluid to remove low-molecular compounds.

Example 1 [Anti-hapten antibody (anti-clenbuterol serum)
Manufacturing method: complete Clenbuterol-HSA 0.5mg
Initial immunization is performed by injecting intradermally into the back of a rabbit together with Freund's adjuvant. 0.2 to 0.5 every two weeks thereafter
mg of protein, and antiserum usable for enzyme immunoassay was obtained 2 to 4 months after the start of immunization.

Example 2 [Production method of clenbuterol - β-D-galactosidase (enzyme-labeled antigen)] Dissolve 0.02 mg of clenbuterol in 0.4 ml of pure water in a small vial, add 0.01 ml of 2NHcl,
Set the pH to 1. Under cover of light, add 0.2 ml of water while stirring while cold.
Slowly drop NaNO ₂ solution (0.1 mg/ml),
After dropping the entire amount, leave it for 30 minutes.

Thereafter, 0.01 ml ammonium sulfonate solution (25 mg/ml) was added to remove free nitrosonium ions.

0.2ml of 0.01M phosphate buffer PH7.5 (0.1M Nacl,
0.1 ml of the diazotized solution of clenbuterol was gradually added to a mixture of 0.05 mg of β-D-galactosidase (E, coli, 470 μ/mg, manufactured by Shikuma) in 1 mM gcl ₂ (containing 1 mM gcl 2 ). Meanwhile, add the solution
Adjust PH7.6 to PH7.7 with 1N NaOH.

After leaving for 2 hours, add 0.2 ml of the above buffer solution to a total volume of 0.5
ml. Immediately, diafiltration was carried out using the above buffer solution as the dialysis solution. After transfection, add this to clenbuterol~
It was stored in the cold as a stock solution of β-D-galactosidase.

Reference Example 2 [Production of carrier adsorbed with second antibody] Goat antibody against rabbit-1gG (1gG fraction of anti-rabbitlgG manufactured by Miles-Yeda)
(Gout)) in 0.05M phosphate buffer (PH7.5, 0.1%)
(contains _NaN3 ), and immerse a polystyrene ball (1/4 inch) into the solution at 4°C.
0.01M phosphate buffer (PH6.6, 0.1% BSA, 0.1M
Contains Nacl and _1mMgcl2 . After washing three times with A1 buffer (hereinafter referred to as _A1 buffer), it was stored cold in the same buffer.

Example 3 (a) [Measurement method] (1) [Sample preparation] Add _A2 buffer solution (0.1M phosphate buffer, PH
Add 0.3ml of 6.6, 0.1M.Nacl, 1mMgcl ₂ ), then add 0.1ml of blood and mix. Heat this small test tube in boiling water for 1 minute. It was immediately cooled in ice water, centrifuged, and the supernatant was used as a sample.

(2) [Method] First, add 0.1 ml of standard clenbuterol (0.125-64 pg) prepared with _A2 buffer or 0.1 ml of sample into a small test tube, and dilute it 8000 times with buffer containing 5% BSA. . Clenbuterol~
Add 0.05 ml of β-D-galactadase. Further, 0.05 ml of antiserum diluted 40,000 times with _A2 buffer containing 5% BSA was added, mixed well, and allowed to stand at 4°C overnight. This was mixed with 0.2 ml of _A2 buffer, and a polystyrene ball (1/4 inch) coated with the second antibody was placed therein, followed by stirring (at room temperature) for 5 hours.

Mix the bowl stirred above with _A1 buffer solution.
Wash twice and pre-transfer the ball to a new test tube. Add 0.2 ml of _A1 buffer to this and add 4-MUG (4-methylumbelliferyl glycoside: 4-MUG), a substrate of β-D-galactosidase.
methylumbelliferyl glucoside) solution 0.2ml (1
mg/10ml water) was added, and the mixture was gently vortexed and incubated at 37°C for 1.5 hours. Enzyme reaction: 2.5ml 0.1M glycine buffer PH
Added 10.3 and stopped it.

Fluorescence intensity is excitation wavelength 360nm, fluorescence wavelength 450nm
Measurements were carried out using a fluorescence spectrophotometer.

(b) [Preparation of calibration curve] A calibration curve as shown in FIG. 1 was prepared using the standard clenbuterol solution, enzyme-labeled antigen, and anti-hapten antibody described in [Measurement method] above. From this calibration curve, the measurable range of the EIA of the present invention is 0.125 ~
64pg/tube.

(c) [Measurement of clenbuterol concentration in blood] Clenbuterol 10 μg to male rats (300 g)
After oral administration, blood was collected from the ear vein over time, and the concentration of clenbuterol in the obtained blood was measured according to the above measurement method (a).

The results are shown in Figure 2.

Example 4 When 20 μg of clenbuterol was orally administered once to three healthy subjects, the time course of clenbuterol concentration in serum was measured by the method of the present invention.

The results are shown in Figure 3.

Example 5 [Preparation of kit for quantifying clenbuterol] Reagents were prepared according to the methods described below, and a kit for measuring clenbuterol by EIA method was prepared.

(a) Enzyme-labeled antigen The stock solution obtained in the above [method for producing clenbuterol-β-D-galactosidase conjugate]
Total volume diluted 10,000 times with _A2 buffer containing % BSA
The enzyme-labeled antigen was prepared by dispensing 2.5 ml into 5 ml glass bottles.

(b) Anti-hapten antibody The anti-hapten antibody obtained in the above [method for producing anti-hapten antibody] was diluted 50,000 times with _A2 buffer containing 5% BSA to make a total volume of 25 ml, and each 2.5 ml was diluted with 5
Anti-hapten antibodies were prepared by dispensing them into ml glass bottles.

(c) Carrier adsorbed with second antibody A goat second antibody against rabbit-IgG was prepared using the same method as described in [Method] of [Measurement method] above.
Antibody (IgG fraction of anti-rabbit I′gG
(Goot)) was adsorbed onto a styrene ball, stored in _A1 buffer solution, and a carrier was prepared by adsorbing the second antibody.

(d) Substrate for measuring enzyme activity 4-, which is a substrate for β-galactosidase
Dissolve 10mg of MUG in 100ml of _A1 buffer and add it to each
A substrate for measuring enzyme activity was prepared by dispensing 10 ml into 20 ml glass bottles.

(e) Clenbuterol standard solution Clenbuterol was dissolved in _A2 buffer containing 5% BSA to obtain 50 ml of a solution with a concentration of 250 pg/ml, and 5 ml of the solution was dispensed into 10 ml bottles.

[Brief explanation of drawings]

Figure 1 shows a calibration curve created using a clenbuterol solution of known concentration, Figure 2 shows the clenbuterol concentration in the blood after oral administration of clenbuterol to male rats, and Figure 3 shows the oral administration of clenbuterol to healthy subjects. This shows the results of measuring clenbuterol concentration in blood by the EIA method of the present invention.

Claims (1)

[Claims] 1. The following formula [] [In the formula, R ₁ and R ₂ represent a chlorine atom or a trifluoromethyl group, and X represents -N ₂ ⁺ . ] An enzyme-labeled antigen obtained by covalently bonding a clenbuterol represented by the following with an enzyme via the group X of the clenbuterol. 2. The enzyme-labeled antigen according to claim 1, wherein in the above formula [], X is _-N2 ⁺ . 3. The enzyme-labeled antigen according to claim 1 or 2, wherein the enzyme is β-D-galactosidase. 4 At least the following reagent (a) the following formula [] [In the formula, R ₁ and R ₂ represent a chlorine atom or a trifluoromethyl group, and X represents -N ₂ ⁺ . ] An enzyme-labeled antigen obtained by covalently bonding a clenbuterol compound represented by the formula [] with an enzyme via the group X of the clenbuterol compound; an anti-hapten antibody obtained by parenterally administering to an animal a hapten antigen obtained by covalently bonding it via a group A kit for enzyme immunoassay of clenbuterol. 5. The kit for enzyme immunoassay of clenbuterols according to claim 4, wherein the carrier to which the second antibody is adsorbed is a styrene ball.