JPH09236603A - Stabilization method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To stabilize a solution containing a stabilized antigen and the antigen by using a water soluble synthetic high polymer compound. SOLUTION: A solution containing an antigen bonding to protein in blood comprises a water soluble synthetic high polymer compound coexisting therein. The water soluble synthetic high polymer compound is made to coexist to obtain a stabilization method of an antigen having a nature of bonding to protein in the blood in the blood. In other words, the water soluble synthetic high polymer compound is used as stabilizing agent to stabilize such an antigen in the solution for a long time. The compound herein used is, for example, preferably polyvinyl alcohol, polyethylene glycol and polyvinyl pyrolidone.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for stabilizing an antigen in a solution.

[0002]

2. Description of the Related Art In recent years, with the development of immunochemistry, the results of immunoassay have been widely used for clinical tests and diagnosis of diseases.

Among various hormones and drugs to be measured by the immunological assay, there are those having a property of binding to proteins in blood, and these have two types in blood, that is, in blood. It exists in a form that is not bound to a protein (free form) and a form that is bound to a protein in blood (bound form). It is also known that some of these have hormonal activity and drug activity only in the free form.

[0004] The binding rate of various hormones and drugs having such properties to proteins in blood changes depending on individual differences and changes in medical conditions. Therefore, specific measurement of the concentrations of these free forms is necessary for accurate diagnosis and selection of appropriate treatment.

Such a free-type measuring method has been developed using an immunological method. In the immunological assay method, a free-form antigen-containing solution (standard substance solution) is usually used to prepare a calibration curve, and this calibration curve is used to measure the free-form concentration of the target substance in various samples. Since such a free standard substance is usually unstable in a solution, it has been conventionally prepared by a method of stabilizing by adding serum itself or serum proteins such as albumin and globulin. However, in the free-form standard substance solution stabilized by adding serum or serum protein, a part of the free-form standard substance is bound to the serum protein in the solution after the preparation to form the bound form, and as a result, There is a problem that the concentration of the standard substance decreases and the concentration of the added free standard substance differs from the concentration of the free standard substance in the prepared standard solution. Moreover, the rate of change of the free standard substance to the bound type changes depending on the type of serum or serum protein, the purity, the production lot, the storage period of the standard substance solution, etc. In order to use in, the concentration of free standard substance must be measured again after the preparation (after mixing the free standard substance and serum protein) at an appropriate time. , Complicated operation was required. Further, even in a standard substance solution in which the concentration of the standard substance is once set, the concentration may change during storage, which causes an analysis error.

Under these circumstances, the development of a method for stabilizing antigens such as various hormones and drugs having a property of binding to proteins in blood without using serum or serum proteins is desired. .

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above situation, and its object is to have a property of binding to proteins in blood stabilized without using serum or serum proteins. An object of the present invention is to provide an antigen-containing solution of various hormones, drugs and the like having the above-mentioned, and a method for stabilizing the antigen.

[0008]

The present invention is an invention of an antigen-containing solution having a property of binding to a protein in blood, which is prepared by allowing a water-soluble synthetic polymer compound to coexist.

The present invention is also an invention of a method for stabilizing an antigen having a property of binding to a protein in blood in a solution, which is characterized in that a water-soluble synthetic polymer compound is allowed to coexist.

That is, the present inventors have earnestly studied about a method of stabilizing an antigen having a property of binding to a protein in blood in a solution without using serum or serum protein,
The present invention has been completed by discovering that such an antigen can be stabilized in a solution for a long period of time by using a water-soluble synthetic polymer compound as a stabilizer.

The water-soluble synthetic polymer compound used in the present invention has a hydrophobic portion and a hydrophilic portion in the molecule,
There is no particular limitation as long as it can be used for the purpose of the present invention, and specifically, for example, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone and the like are preferable. The weight average molecular weight of these water-soluble synthetic polymer compounds is usually 1000 to 250,000,
It is preferably about 3,000 to 100,000, and the concentration used as a stabilizer in a solution containing an antigen to be stabilized varies slightly depending on the type of water-soluble synthetic polymer compound used. , Usually 0.01-10 (W / V)%,
Preferably, it is appropriately selected from the range of 0.01 to 5 (W / V)%.

As a preferred specific example of polyvinyl alcohol used as the water-soluble synthetic polymer compound of the present invention, an average molecular weight is usually 4400 to 220000 (average polymerization degree of 100 to 5).
000), preferably those having an average molecular weight of 8800 to 132000 (average polymerization degree of 200 to 3000). When polyvinyl alcohol is used as a stabilizer in an antigen-containing solution, the concentration is usually 0.01 to 5 (W / V)%, preferably 0.1 to 2
(W / V)%. It should be noted that polyvinyl alcohol has a lower solubility in water as the molecular weight increases, and it becomes 5 (W / V).
In some cases, it may dissolve only to a concentration less than%,
In such a case, the concentration naturally becomes the upper limit of the concentration during use. In addition, when the polyvinyl alcohol concentration exceeds 2 (W / V)%, the viscosity of the solution becomes high, so the precision during dispensing may decrease, in which case errors in analysis will easily occur. It is better to use it not to exceed 2 (W / V)%. Further, the polyvinyl alcohol used in the present invention may be one which has not been completely saponified as long as it is soluble in water, the proportion of the saponification is not particularly limited, but usually 70% of the whole. Above, preferably 80% or more saponified is used. Further, as a preferred specific example of polyethylene glycol used as the water-soluble polymer compound of the present invention, its weight average molecular weight is usually 1000 to 250,000, preferably 3000.
~ 100000, as a preferred specific example of polyvinylpyrrolidone, its weight average molecular weight is usually 1000
˜250,000, preferably 3,000 to 100,000. When polyethylene glycol or polyvinylpyrrolidone is used as a stabilizer in an antibody-containing solution, its concentration is usually 0.01 to 10 (W / V)%, preferably 0.01 to 5 (W
/ V)%.

Antigens having the property of binding to proteins in blood, which can be stabilized by the present invention, are classified into two types in blood,
That is, it exists in a form that is not bound to a protein in blood (free form) and a form that is bound to a protein in blood (bound form). So-called antigen or hapten may be used as long as physiological activity such as activity and drug activity is recognized, and is not particularly limited. Specifically, a protein having the above-mentioned properties,
Measurement of free concentrations of peptides, nucleic acids, sugar chains, lipids, hormones, drugs, etc. in biological fluids such as serum, blood, plasma, etc. in biological samples such as urine, lymphocytes, blood cells, various cells All such values are clinically useful. More specifically, the binding rate to proteins in blood is usually 5 to less than 100%, preferably less than 30 to 100%, more preferably less than 70 to 100%, still more preferably less than 80 to 100%. Preferred haptens are mentioned. Specific examples of the hapten include phenytoin, carbamazepine,
Antiepileptic drugs such as valproic acid, phenobarbital, primidone, clonazepam, diazepam, and nitrazepam,
For example, antipsychotics such as chlorpromazine and haloperidol, antidepressants such as imipramine, desipramine, amitriptyline, nortriptyline, bronchodilators such as theophylline, cardiac glycosides such as digoxin and digitoxin, antiarrhythmic drugs such as quinidine. , For example, aminoglycoside antibiotics, antibacterial agents such as chloramphenicol, anticancer agents such as methotrexate, immunosuppressants such as cyclosporine, drugs such as antipyretic analgesics such as aspirin, such as thyroxine (T4), avian Thyroid hormones such as iodothyronine (T3), and hormones such as adrenocortical hormones such as testosterone and corticosterone are preferred. Among them, it is particularly effective for stabilizing the following haptens in a solution in which the binding rate to proteins in blood is less than 70 to 100%. That is, thyroid hormones such as thyroxine (T4) and triiodothyronine (T3), adrenocortical hormones such as testosterone and corticosterone, phenytoin, diazepam, chlorpromazine, imipramine, desipramine, amitriptyline, nortriptyline, digitoxin and quinidine. .

The binding rate of an antigen to a protein in blood used herein is the equilibrium dialysis method ("Chemistry and Pharmacy", which is a measurement method usually used in this field).
Volume 70, pp. 6-7 (1981), published by Hirokawa Shoten Co., Ltd.).

To carry out the present invention, for example, the following may be carried out. That is, in a suitable solution, the target antigen to be stabilized has a predetermined concentration, and the water-soluble synthetic polymer compound according to the present invention is usually 0.01 to 10 (W / V)%, preferably 0.1 to 5 (W
/ V)% so that it can be carried out by dissolving.

If an antigen solution is prepared according to the present invention,
An antigen solution having a desired concentration can be easily obtained, and it is not particularly necessary to measure the antigen concentration again, but the antigen concentration may be measured in order to confirm the antigen concentration after preparation.

The order of dissolution of the antigen and the water-soluble synthetic polymer compound is not particularly limited, but it is possible to further reduce the fluctuation of the antigen concentration by first dissolving the water-soluble synthetic polymer compound and then dissolving the antigen. So desirable.

The solution in which the antigen and the water-soluble synthetic polymer compound are dissolved is not particularly limited as long as it does not lose the properties of the antigen. Usually, for example, those used as buffers in immunoassays are preferably used. Specifically, for example, phosphates, acetates, citrates, Good
Buffers such as tris (hydroxyethyl) aminomethane, salts such as sodium chloride, potassium chloride and ammonium sulfate, organic solvents such as methanol, ethanol, isopropanol, acetonitrile and tetrahydrofuran, such as polyoxyethylene (10 )
Usually, a buffer solution having a pH of 2 to 11, preferably a pH of 4 to 9, which is prepared by appropriately selecting from surfactants such as octyl phenyl ether and polyoxyethylene higher alcohol ether according to the properties of the antigen, is preferably used.

The stabilized antigen-containing solution of the present invention is an antigen having a property of binding to a protein in blood and has two types in blood, that is, a type not bound to a protein in blood (free Type) and the type bound to the protein in the blood (bound type)
It is useful as a standard solution in the case of trying to measure an antigen which is present in the free form and whose physiological activity such as hormonal activity and drug activity is observed only in its free form by a so-called immunological assay method. It has high quality. Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0020]

【Example】

Embodiment 1 FIG. Study on stabilization of thyroxine (T4) standard solution (buffer solution for T4 standard solution preparation) Good buffer MES (2-M
21.3g of orpholinoethanesulfonic acid, monohydrate) is dissolved in 900ml of ion-exchanged water and adjusted to pH 7.0 with 1N NaO.
A buffer solution for preparing a T4 standard solution was prepared by adding an aqueous solution of H and making the total amount 1 liter. (T4 standard solution) To the T4 standard solution preparation buffer prepared above,
Polyvinyl alcohol (average degree of polymerization: 200 to 230, Aldrich, average molecular weight: 880 to 10120), polyethylene glycol 4000 (Wako Pure Chemical Industries, Ltd., average molecular weight: 4000),
Polyvinylpyrrolidone (Wako Pure Chemical Industries, Ltd., average molecular weight 40,000), bovine serum albumin (Sigma) or bovine globulin (Sigma) 1 (W / V)% and T4 (Sigma) respectively Was dissolved at a concentration of 1 ng / dl to prepare a T4 standard solution. As a control, a T4 standard solution was prepared by simply dissolving T4 in the above-prepared T4 standard solution preparation buffer at 1 ng / dl. (Antibody solution) Anti-T4 monoclonal antibody (Capricorn)
Was treated with a conventional method to obtain Fab ', and this was labeled with horseradish peroxidase (POD) by a conventional method to obtain PO.
Antibodies were prepared by adding D-labeled anti-T4-Fab '(hereinafter Fab'-POD) to 0.2 M MES buffer (pH 7.0) so that Fab'-POD had a concentration of 9.3 × 10 ^-10 M. It was a liquid. [Analysis conditions by high performance liquid chromatography (HPLC)] The outline of the system is shown in FIG. The measurement conditions are as follows.・ Column: POROS-S (4.6φ × 35 mm, manufactured by Perseptive) ・ Eluent: A solution: 20 mM MES buffer (pH 6.5) B solution: A solution containing NaCl at 1 M : 2 ml / min-Substrate solution: 50 mM citrate buffer (pH 5.5) containing 300 mM acetamide phenol and 40 mM hydrogen peroxide. Flow rate: 0.2 ml / min ・ Reaction part: 0.25 mmφ × 10 m coil (60 ° C heat retention) ・ Detection: Excitation wavelength 328 nm, fluorescence wavelength 432 nm. Gradient conditions: 0 → 3 min B = 0 → 20% 3 → 3.5 min B = 100% (Measurement procedure) 100 μl of antibody solution and 10 μl of T4 standard solution were mixed and allowed to stand at 8 ° C. for 2.5 minutes. 50 μl HPLC
Was analyzed by (Results) Table 1 shows the measurement results of the free T4 concentration of the prepared T4 standard solution immediately after preparation, after storage at 30 ° C for 1 week, and after storage at 30 ° C for 3 weeks.

[0021]

[Table 1]

From the results shown in Table 1, the T4 standard solution in which polyvinyl alcohol, polyethylene glycol and polyvinyl pyrrolidone, which are water-soluble polymer compounds according to the present invention, coexist can store free T4 stably for a long period of time. ,
Further, it is found that even when albumin or globulin, which is a conventional stabilizer, is allowed to coexist, the T4 concentration of the free form is remarkably lowered immediately after preparation, and further, the T4 concentration of the free form fluctuates with time.

Embodiment 2 FIG. Examination of Stabilization of Thyroxine Solution by Polyvinyl Alcohol (T4 Standard Solution) Polyvinyl alcohol having a predetermined average degree of polymerization was dissolved in the T4 standard solution preparing buffer prepared in Example 1 to a predetermined concentration, and then T4 ( Sigma) 1 ng /
The T4 standard solution was also prepared by dissolving it to give dl. still,
The polyvinyl alcohol used is as follows. a) Average degree of polymerization 200 to 230 (Aldrich, saponification degree 80%), b) Average degree of polymerization 500 (Wako Pure Chemical Industries, Ltd., saponification degree 96% or more), c) Average degree of polymerization 1500 (Wako Pure) Pharmaceutical Industry Co., Ltd.), d) Average degree of polymerization 2800 (Wako Pure Chemical Industries Ltd., saponification degree 86-90%)
, E) Average degree of polymerization 3500 (Wako Pure Chemical Industries, Ltd., saponification degree
86-90%). (Antibody solution) The same as in Example 1. (Analysis conditions by HPLC) The same as in Example 1. (Measurement operation) The same operation as in Example 1 was performed. (Results) Table 2 shows the results of measuring the free T4 concentration of the prepared T4 standard solution after storage at 30 ° C for 1 week.

[0024]

[Table 2] *: Wako Pure Chemical Industries, Ltd.

From the results shown in Table 2, even if the average degree of polymerization and the concentration of polyvinyl alcohol are changed, the T4 stabilizing ability of the polyvinyl alcohol is not changed. In other words, the T4 stabilizing ability of polyvinyl alcohol is not changed. It can be seen that the average degree of polymerization and the concentration used do not change.

[0026]

Industrial Applicability As described above, the present invention provides an antigen-containing solution stabilized by using a water-soluble synthetic polymer compound, and a method for stabilizing an antigen. Antigens that have the property of binding to proteins in blood exist in two types in blood, namely, types that are not bound to proteins in blood (free form) and types that are bound to proteins in blood (bound form). However, according to the present invention, the free form of the antigen, in which only the free form of the two forms has physiological activity such as hormone activity, drug activity, etc., is stably dissolved in the free form for a long period of time. Can be stored in. Therefore, if the antigen-containing solution stabilized by the present invention is used as a standard solution,
The present invention is a great invention that contributes to the related art, because it has the effect of allowing free-form measurement to be performed more accurately than when using a conventional standard solution.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a high performance liquid chromatograph used in Examples 1 and 2.

Claims (4)

[Claims] 1. An antigen-containing solution having a property of binding to a protein in blood, which is prepared by allowing a water-soluble synthetic polymer compound to coexist. 2. The solution according to claim 1, wherein the antigen is a hapten. 3. The solution according to claim 2, wherein the hapten has a binding rate to a protein in blood of 70% or more. 4. A method for stabilizing an antigen having a property of binding to a protein in blood in a solution, which comprises allowing a water-soluble synthetic polymer compound to coexist.