JP2018163149A - Manufacturing method for antigen-containing solution and immunoassay method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manufacturing method for an antigen-containing solution in which an antigen hardly changes in activity, and an immunoassay method capable of determining the quantity of an antigen in a sample accurately with high reproducibility.SOLUTION: A manufacturing method for antigen-containing solution includes a process (I) of aging a solution (X) containing an antigen (A) and a chelate agent (B) at 15-50°C, 1≤S≤-9.57×T+508.6 (1) holding in the process (I) for an aging temperature T(°C) and an aging time S(day).SELECTED DRAWING: None

Description

  The present invention relates to a method for producing an antigen-containing aqueous solution and an immunoassay method.

Antigens (especially cytokeratin 19 fragments) are often used for diagnosis of diseases and monitoring of treatment as tumor markers highly specific for non-small cell lung cancer, particularly squamous cell carcinoma. In order to quantify antigens (especially cytokeratin 19 fragments) in patient samples, a calibrator is required as a reference standard. However, since the antigen contained in this calibrator is dilute, it is difficult to stably store it in a solution for a long time. Therefore, it is stored in a form in which water has been removed by lyophilization and dissolved by adding water at the time of use, a method of storing in liquid form at -20 ° C. in the presence of 50% by weight of glycerol to prevent freezing denaturation, and bovine serum albumin A method for securing stability by adding a protein such as animal serum to freeze an aqueous solution is known (for example, see Non-Patent Documents 1 and 2).
However, in the lyophilization method, it is necessary to dissolve the solution to a required concentration with a buffer solution or the like before use, and an error is likely to occur in the amount of the solution to be dissolved, which affects the accuracy in quantifying patient samples. is there. Further, the method of liquid storage at −20 ° C. in the presence of 50% by weight of glycerol has a problem that it cannot be directly used for immunoassay as a calibration substance because of its high viscosity and low sampling accuracy. Furthermore, in the method of freezing an aqueous solution by adding a protein such as bovine serum albumin or animal serum, there is a problem that accuracy cannot be ensured due to the effect of inactivation due to freezing and thawing of an antigen (particularly cytokeratin 19 fragment).

Atsushi Sasaki, Immunochemical Identification Method (3rd edition), Tokyo Chemical Doujin, 1993 Keiji Mizuoka, Immunoserum Test Manual, Kodansha Scientific, 1986

  An object of the present invention is to provide a method for producing an antigen-containing aqueous solution in which the activity of an antigen hardly changes, and an immunoassay method capable of accurately and reproducibly quantifying an antigen in a sample.

The inventor of the present invention has arrived at the present invention as a result of intensive investigations to achieve the above object.
That is, the present invention includes the step (I) of aging the aqueous solution (X) containing the antigen (A) and the chelating agent (B) at 15 to 50 ° C., and the aging temperature T (° C.) in the step (I) A method for producing an antigen-containing aqueous solution that satisfies the relationship of the following mathematical formula (1) with the aging time S (day); a step of preparing a calibration curve using the antigen-containing aqueous solution obtained by this production method as a known amount of the specimen It is an immunoassay method including.
1 ≦ S ≦ −9.57 × T + 508.6 (1)

According to the method for producing an antigen-containing aqueous solution of the present invention, an antigen-containing aqueous solution in which the activity of the antigen is hardly changed can be obtained.
Moreover, the immunoassay method of the present invention can quantify the antigen in the sample accurately and with high reproducibility.

The method for producing an antigen-containing aqueous solution of the present invention includes the step (I) of aging an aqueous solution (X) containing the antigen (A) and the chelating agent (B) at 15 to 50 ° C. ) In which the aging temperature T (° C.) and the aging time S (day) satisfy the relationship of the following mathematical formula (1).
1 ≦ S ≦ −9.57 × T + 508.6 (1)
According to the production method of the present invention, the activity of the antigen (A) is changed by aging the aqueous solution (X) containing the antigen (A) and the chelating agent (B) at 15 to 50 ° C. for the aging time. It becomes difficult. Therefore, if the antigen-containing aqueous solution obtained by the production method of the present invention is used as, for example, a calibration substance in immunoassay (a known amount of analyte used in preparing a calibration curve), the antigen in the patient sample can be accurately obtained. In addition, it can be quantified with high reproducibility. In addition, the manufacturing method of this invention should just contain the said process (I), and the obtained antigen containing aqueous solution may further be stored at refrigeration (4-10 degreeC) or room temperature (10-35 degreeC). .

In the present invention, the antigen (A) includes a cytokeratin 19 fragment.
The cytokeratin 19 fragment is a soluble fragment of cytokeratin 19 that is one of cytokeratins that are constituent proteins of intermediate filaments of epithelial cells, and is an acidic protein having a molecular weight of about 40 kD.
The origin of the cytokeratin 19 fragment used in the present invention is not particularly limited, and a genetically modified product or a chemically synthesized product can be used in addition to the natural product extracted from the tissue. These antigens are commercially available from Roche and Biodesign International.

  In the present invention, the content of the antigen (A) in the aqueous solution (X) can be used at any concentration, but it is necessary for quantifying the antigen (particularly the cytokeratin 19 fragment) in the blood of the patient. From the viewpoint of the concentration range and the viewpoint that the activity of the antigen (A) is less likely to change, it is preferably 1 to 1000 μg / L, more preferably 2 to 100 μg / L, and still more preferably, based on the volume of the aqueous solution (X). Is 3 to 10 μg / L.

In the present invention, the chelating agent (B) includes a compound having a total of at least two functional groups selected from the group consisting of an amino group, a hydroxyl group and a carboxyl group, such as an aminocarboxylic acid type chelate. Agents and hydroxy acid type chelating agents.
Examples of aminocarboxylic acid type chelating agents include ethylenediaminetetraacetic acid (hereinafter also referred to as “EDTA”), N- (2-acetamido) iminodiacetic acid (hereinafter also referred to as “ADA”), glycol etherdiaminetetraacetic acid, Examples include diethylenetriaminepentaacetic acid, ethylenediamine-N, N′-disuccinic acid and 3-sodium-2,2′-iminodisuccinic acid tetrasodium, and salts thereof.
Examples of the hydroxy acid type chelating agent include α-hydroxy acids (for example, glycolic acid, lactic acid, tartaric acid, malic acid, citric acid and the like), β-hydroxy acids (for example, salicylic acid and the like), and salts thereof.
Examples of the salt include alkali metal salts (for example, lithium salt, sodium salt and potassium salt), alkaline earth metal salts (for example, magnesium salt and calcium salt) and the like.
The chelating agent (B) in this invention may be used individually by 1 type, or may use 2 or more types together.

  As the chelating agent (B), from the viewpoint that the activity of the antigen (A) is less likely to change, from ethylenediaminetetraacetic acid, N- (2-acetamido) iminodiacetic acid, citric acid, malic acid, gluconic acid and salts thereof At least one selected from the group consisting of

  The chelating agent (B) in the present invention can be contained in combination with other purposes such as a buffering agent in addition to the additive for improving the stability.

  The content of the chelating agent (B) in the aqueous solution (X) is preferably 0.1 to 100 g / L based on the volume of the aqueous solution (X) from the viewpoint that the activity of the antigen (A) is difficult to change. More preferably, it is 1-50 g / L, More preferably, it is 10-30 g / L.

In the production method of the present invention, the aging temperature is 15 ° C. to 50 ° C., and preferably 20 ° C. to 40 ° C., particularly preferably 25 ° C. to 30 ° C. from the viewpoint that the activity of the antigen (A) is less likely to change. It is.
If the aging temperature is less than 15 ° C., a sufficient stabilizing effect cannot be obtained. On the other hand, when the aging temperature is higher than 50 ° C., the activity of the antigen decreases.

In step (I) of the production method of the present invention, the aging temperature T (° C.) and the aging time S (day) satisfy the relationship represented by the following mathematical formula (1).
1 ≦ S ≦ −9.57 × T + 508.6 (1)
The above formula (1) represents the relationship between the aging temperature T and the aging time S in the step (I), and means that the higher the aging temperature T, the shorter the aging time S. By satisfying 1), a sufficient stabilizing effect by the chelating agent (B) can be obtained, and an antigen-containing aqueous solution that hardly changes the activity of the antigen (A) can be produced.

  From the viewpoint of the storage stability of the antigen (A) in the antigen-containing aqueous solution, the pH of the aqueous solution (X) and the antigen-containing aqueous solution of the present invention is preferably 5.5 to 8.5, more preferably 6.0 to 7 .8, particularly preferably 6.5 to 7.0. When the pH is 5.5 or more or the pH is 8.5 or less, the storage stability of the antigen (A) is high, and the activity of the antigen (A) is hardly changed.

In the production method of the present invention, the aqueous solution (X) and the antigen-containing aqueous solution may contain a buffer, a preservative, and other stabilizers in addition to the antigen (A) and the chelating agent (B).
Examples of the buffer include a phosphate buffer and a good buffer.
Examples of preservatives include sodium azide and methylisothiazolinone.
Examples of other stabilizers include bovine serum albumin, trehalose and arginine.

  Since the antigen-containing aqueous solution obtained by the production method of the present invention does not easily change the activity of the antigen (A) in the antigen-containing aqueous solution, for example, a calibration curve is created using a known amount of the analyte and the measurement value is calibrated. It can be used as a calibrator in immunoassay methods that need to be performed.

  The immunoassay method of the present invention includes a step of preparing a calibration curve using the antigen-containing aqueous solution obtained by the above production method as a known amount of a specimen (hereinafter also referred to as a calibration substance).

The immunoassay method in the present invention includes the above steps, and is not particularly limited as long as it is generally measured in this field. For example, chemiluminescent enzyme immunoassay (CLEIA), enzyme immunoassay (EIA), radioimmunoassay (RIA), electrochemiluminescence immunoassay (ECLIA), fluorescent enzyme immunoassay (FEIA), chemiluminescence immunoassay (CLIA), latex optical immunoassay (LPIA) and Examples thereof include a latex particle counting immunoagglutination assay (CIA) and the like.
In these measurement methods, the antigen-containing aqueous solution of the present invention is preferably used as it is as a calibration substance.

  Furthermore, when the immunoassay is a chemiluminescent enzyme immunoassay, the solid phase carrier is not particularly limited as long as it is generally measured in this field. For example, glass beads, polystyrene beads, magnetic silica particles Typical examples include microplates and latex. Specifically, for example, known magnetic silica particles described in JP 2014-210680 A and JP 2013-019889 A are preferable.

  The magnetic silica particles in the chemiluminescent enzyme immunoassay include those in which a metal oxide having an average particle diameter of 1 to 15 nm and having superparamagnetism is dispersed in a silica matrix. Superparamagnetism refers to a temporary magnetic field that is induced by the individual atomic magnetic moments of a material being aligned in the presence of an external magnetic field, and that when the external magnetic field is removed, partial alignment is impaired and no magnetic field is exhibited. means.

  Examples of superparamagnetic metal oxides having an average particle diameter of 1 to 15 nm and exhibiting superparamagnetism include oxides such as iron, cobalt, nickel, and alloys thereof. Iron is particularly preferred. A superparamagnetic metal oxide may be used individually by 1 type, or may use 2 or more types together.

  The lower limit of the content of superparamagnetic metal oxide in the magnetic silica particles is preferably 60% by weight, more preferably 65% by weight, and the upper limit is preferably 95% by weight, more preferably 80% by weight. When the content of the superparamagnetic metal oxide is 60% by weight or more, the magnetic silica particles obtained have sufficient magnetism, and the separation operation in actual use can be performed in a short time, and is 95% by weight or less. Easy to manufacture.

  The aqueous solution containing the antigen (A) is generally stored frozen because it is gradually inactivated by refrigeration (about 4 ° C to 10 ° C) storage or storage at room temperature (10 to 35 ° C). There is a problem that it is deactivated by repeating. On the other hand, in the present invention, by aging the aqueous solution (X) containing the antigen (A) and the chelating agent (B) at 15 to 50 ° C., the activity of the antigen (A) becomes difficult to change, and the antigen ( A) refrigeration or long-term storage at room temperature is possible.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these.

<Example 1>
[Preparation of aqueous solution containing antigen]
0.02M phosphate buffer (pH 7.0) containing 0.1% by weight bovine serum albumin, 0.85% by weight sodium chloride and 20 g / L EDTA in a room temperature-controlled at 25 ° C. 25 ° C.).
Cytokeratin 19 fragment antigen (Merdian Life Science Co., Ltd., “Cyfra-21-1 Antigen”) was added to 5 μg / L, and then matured at 25 ° C. for 14 days to give the antigen-containing aqueous solution 1 Obtained.

<Examples 2 to 5>
In Example 1, antigen-containing aqueous solutions 2 to 5 were obtained in the same manner except that the chelating agents shown in Table 1 were used instead of “EDTA”.

<Examples 6 to 7>
In Example 1, antigen-containing aqueous solutions 6 to 7 were obtained in the same manner except that the aging conditions shown in Table 1 were used.

<Examples 8 to 9>
In Example 1, antigen-containing aqueous solutions 8 to 9 were obtained in the same manner except that the pH was adjusted to the values shown in Table 1.

<Examples 10 to 11>
In Example 1, antigen-containing aqueous solutions 10 to 11 were obtained in the same manner except that the antigen concentrations shown in Table 1 were prepared.

<Examples 12 to 13>
In Example 1, antigen-containing aqueous solutions 12 to 13 were obtained in the same manner except that the chelating agent concentrations shown in Table 1 were prepared.

<Comparative Example 1>
A comparative antigen-containing aqueous solution 1 ′ was obtained in the same manner as in Example 1 except that “EDTA” was not used.

<Comparative example 2>
In Example 1, the cytokeratin 19 fragment antigen was added to 5 μg / L, and the one not matured was used as a comparative antigen-containing aqueous solution 2 ′. In the “Evaluation of Storage Stability of Antigen-Containing Aqueous Solution” to be described later, the product at the time of preparation was 5 hours (25 ° C.) after adding the antigen.

<Comparative Example 3>
A comparative antigen-containing aqueous solution 3 ′ was obtained in the same manner as in Comparative Example 2 except that “EDTA” was not used.

<Comparative example 4>
A comparative antigen-containing aqueous solution 1 ′ was obtained in the same manner as in Example 1 except that the aging temperature was 10 ° C.

[Evaluation of storage stability of antigen-containing aqueous solution]
The antigen-containing aqueous solutions prepared in Examples 1 to 13 and Comparative Examples 1 to 4 were stored at 4 ° C. and 35 ° C., respectively, and at the time of preparation, cytokeratin 19 at the second week, the first month, the second month, and the third month The fragment concentration was measured with a cytokeratin 19 fragment measurement reagent (Roche Diagnostics Co., Ltd., “Eccursis reagent”), and the ratio (%) to the concentration at the time of preparation {(concentration obtained by measurement) / ( (Concentration at the time of production) × 100} was calculated. The results are shown in Table 1. In addition, it shows that the activity of an antigen is hard to change, so that the ratio with respect to the density | concentration at the time of preparation is near 100%.

From the results of Table 1, the antigen-containing aqueous solutions obtained in Examples 1 to 13 have a ratio of 95% to 105% with respect to the concentration at the time of preparation when the storage temperature is 4 ° C, and the storage temperature is 35 ° C. In this case, the ratio to the concentration at the time of preparation is in the range of 93% to 115%, and it can be seen that the activity of the antigen hardly changes and is hardly affected by temperature.
On the other hand, the antigen-containing aqueous solutions obtained in Comparative Examples 1 to 4 have a ratio of 93% to 109% with respect to the concentration at the time of preparation when the storage temperature is 4 ° C. It can be seen that the change is large. In addition, when the storage temperature is 35 ° C., it is 144% to 214%, which indicates that the activity of the antigen is easily changed, and further, the activity of the antigen is easily changed by the influence of the temperature.
Therefore, it can be seen that the antigen-containing aqueous solution of the present invention hardly changes the antigen activity regardless of the storage temperature. In addition, if a calibration curve is created using the antigen-containing aqueous solution of the present invention as a known amount of analyte, a highly accurate calibration curve can be created, so that the antigen in the sample can be quantified accurately and with high reproducibility. You can see that

  The antigen-containing aqueous solution prepared by the method of the present invention can be stably stored for a long time in a solution state. Therefore, by using the antigen-containing aqueous solution of the present invention as a calibration substance, immunoassay with good accuracy and reproducibility can be performed, and the reliability of clinical tests can be enhanced.

Claims (6)

Including the step (I) of aging the aqueous solution (X) containing the antigen (A) and the chelating agent (B) at 15 to 50 ° C. In the step (I), the aging temperature T (° C.) and the aging time S (days) And a method for producing an antigen-containing aqueous solution satisfying the relationship of the following mathematical formula (1).
1 ≦ S ≦ −9.57 × T + 508.6 (1)   The method for producing an antigen-containing aqueous solution according to claim 1, wherein the antigen (A) is a cytokeratin 19 fragment.   The chelating agent (B) is at least one selected from the group consisting of ethylenediaminetetraacetic acid, N- (2-acetamido) iminodiacetic acid, citric acid, malic acid, gluconic acid, and salts thereof. A method for producing the antigen-containing aqueous solution according to 1.   The method for producing an antigen-containing aqueous solution according to any one of claims 1 to 3, wherein the pH of the aqueous solution (X) is 5.5 to 8.5.   The concentration of the antigen (A) is 1-1000 μg / L and the concentration of the chelating agent (B) is 0.1-100 g / L based on the volume of the aqueous solution (X). 2. A method for producing an antigen-containing aqueous solution according to item 1.   An immunoassay method comprising a step of creating a calibration curve using the antigen-containing aqueous solution obtained by the production method according to any one of claims 1 to 5 as a known amount of a subject.