JP4568334B2 - Antigen-containing aqueous solution - Google Patents

Description

  The present invention relates to an antigen-containing aqueous solution that can prevent inactivation of an antigen in a solution containing the antigen and can be stably stored.

When performing an immunoassay, a standard solution containing an antigen is generally used as a calibrator. Usually, since the antigen contained in this standard solution is dilute, some antigen species may be remarkably inactivated in the solution. In particular, antigen solutions such as hormones and enzymes are highly inactivated and are often difficult to use as an aqueous solution calibrator. In such a case, store it in a form that has been dehydrated by a method such as lyophilization and add water at the time of use to dissolve this individual, or add a protein such as bovine serum albumin or animal serum to an aqueous solution. Measures have been taken to ensure the stability inside (Non-Patent Documents 1 and 2).
Atsushi Sasaki, Immunochemical identification method (3rd edition), Tokyo Chemical Doujin, 1993 Ishikawa, Yuji, Ultrasensitive Enzyme Immunoassay, Academic Publishing Center, 1993

  However, the conventional freeze-drying method has good storage stability, but water must be added at the time of use, so that the operation becomes complicated, and as a result, an error is likely to occur in the antigen concentration in the standard solution. In particular, when an N-terminal brain natriuretic peptide precursor aqueous solution is used as a standard solution, the stability of the N-terminal brain natriuretic peptide precursor in the solution can be improved even when proteins such as bovine serum albumin and animal serum are added. It is difficult to ensure long-term storage stability. That is, an object of the present invention is to provide a composition capable of stably storing an N-terminal brain natriuretic peptide precursor in an aqueous solution.

  The antigen-containing aqueous solution of the present invention is characterized by containing an N-terminal brain natriuretic peptide precursor, casein and / or whey protein, and at least one functional group selected from the group consisting of an amide group, a sulfo group and a carboxyl group And having a pH of 5.5 to 7.0.

The antigen-containing aqueous solution of the present invention has the following effects.
(1) The N-terminal brain natriuretic peptide precursor which is easily inactivated can be stably stored for a long time in a solution state.
(2) The stability can be maintained for a long time without lyophilization performed in the past, and there is no concentration error caused by a complicated preparation operation of dissolving the antigen, and immunoassay with good measurement accuracy and reproducibility is achieved. it can.

The human-derived N-terminal brain natriuretic peptide precursor is a degradation product of natriuretic peptide precursor (proBNP) synthesized in cardiomyocytes and is composed of 76 amino acids.
The origin of the N-terminal cerebral natriuretic peptide precursor 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 a natural product extracted from a tissue. These antigens are commercially available from HyTest, Immunodiagnostic AG, and the like.

  The content of the N-terminal brain natriuretic peptide precursor can be used at any concentration as long as it is 10 mg / g or less based on the weight of the antigen-containing aqueous solution, but the N-terminal brain natriuretic peptide precursor in the blood can be used. From the viewpoint of the concentration range required for quantifying the body, 10 pg / g to 1 μg / g is preferable, more preferably 100 pg / g to 100 ng / g, and still more preferably 1 ng, based on the weight of the antigen-containing aqueous solution. / G to 10 ng / g.

  Casein includes casein in milk. For example, casein described in “Fifth Edition Food Additives Official Document (published by Yodogawa Shoten)” can be used. Specific examples include α-casein, β-casein, γ-casein, κ-casein, and λ-casein.

  From the viewpoint of storage stability of the N-terminal brain natriuretic peptide precursor in the antigen-containing aqueous solution, the concentration of casein is preferably 0.01% to 3%, more preferably 0.00%, based on the weight of the antigen-containing aqueous solution. 1 to 2.5%, then more preferably 0.5 to 2%.

  Whey protein includes whey protein in milk. For example, concentrated whey and whey powder described in the “Ministry Ordinance on Component Standards of Milk and Dairy Products” can be used.

  From the viewpoint of storage stability of the N-terminal brain natriuretic peptide precursor in the antigen-containing aqueous solution, the concentration of the whey protein is preferably 0.01% to 3%, more preferably 0, based on the weight of the antigen-containing aqueous solution. .1 to 2.5%, and further preferably 0.5 to 2%.

  In the antigen-containing aqueous solution of the present invention, casein and whey protein can be used alone, but casein and whey protein can also be used in combination. When used together, the total concentration of casein and whey protein is 0.01% to the weight of the antigen-containing aqueous solution from the viewpoint of the storage stability of the N-terminal brain natriuretic peptide precursor in the antigen-containing aqueous solution. 3% is preferable, more preferably 0.1 to 2.5%, and still more preferably 0.5 to 2%.

  The coexistence of casein and whey protein can further improve the storage stability of an aqueous solution containing an N-terminal brain natriuretic peptide precursor. The concentrations at this time are preferably 0.5% to 1.25% casein and 0.5% to 1.25% whey protein, particularly preferably 0.25% to 0.63% casein, 0. 25% to 0.63%.

The antigen-containing aqueous solution of the present invention has a pH of 5.5 to 7.0, and preferably 5.8 to 6.9 from the viewpoint of storage stability of the N-terminal brain natriuretic peptide precursor in the antigen-containing aqueous solution. More preferably, it is 6.1 to 6.8.
When the pH is less than 5.5, the stability of casein and / or whey protein is deteriorated, and when the pH is more than 7.0, the storage stability of the N-terminal brain natriuretic peptide precursor is deteriorated.
In addition, pH can be measured by JIS K0400-12-10: 2000 (measurement temperature 25 degreeC).

In order to adjust the pH of the antigen-containing aqueous solution within the above range, the pH can be adjusted using a buffer described later.
The pH is adjusted by using an inorganic alkali hydroxide in the buffer solution in the same manner as a general method for preparing a buffer solution. Examples of the inorganic alkali hydroxide include sodium hydroxide, potassium hydroxide, and lithium hydroxide.

  As the buffer contained in the antigen-containing aqueous solution of the present invention, a buffer known as Good buffer is preferably used. Among the Good buffers, a buffer having a buffer capacity between pH 5.5 and 7.0 is preferable from the viewpoint of storage stability.

From the viewpoint of antigen stability in an aqueous solution, the buffer contains a buffer containing at least one functional group selected from the group consisting of an amide group, a sulfo group and a carboxyl group.
Two or more types of buffering agents may be used in combination, but it is preferable to use one type from the viewpoint of antigen stability in an aqueous solution.

  As the buffer containing an amide group, N- (2-acetamido) iminodiacetic acid (hereinafter abbreviated as ADA. The notation in the parentheses of the compound name described later similarly represents an abbreviation), N- (2-acetamido ) -2-aminoethanesulfonic acid (ACES) and the like.

  Examples of the buffer containing a sulfo group include 2-morpholinoethanesulfonic acid monohydrate (MES), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), N- (2-acetamido)- 2-aminoethanesulfonic acid (ACES), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 3-morpholinopropane Sulfonic acid (MOPS) and the like are included.

  Examples of the buffer containing a carboxyl group include N- (2-acetamido) iminodiacetic acid (ADA).

  Of these, ADA, PIPES, and ACES are preferable from the viewpoint of antigen stability in an aqueous solution, more preferably ADA, ACES, and still more preferably ADA.

The antigen-containing aqueous solution of the present invention can be obtained by mixing an N-terminal brain natriuretic peptide precursor, casein and whey protein, and a buffer.
As a mixing method, the same method as that for producing a conventional antigen-containing aqueous solution may be used.
The antigen-containing aqueous solution of the present invention is prepared by adding casein and / or whey protein after preparing a buffer solution from the viewpoint of ease of production and prevention of inactivation of the N-terminal brain natriuretic peptide precursor at the time of production. The N-terminal brain natriuretic peptide precursor is then added and mixed.

  The antigen-containing aqueous solution of the present invention can be used when performing immunoassay such as enzyme immunoassay, radioimmunoassay, or immunoturbidimetry in the field of clinical examination. In particular, it can be suitably used as a standard solution because it can prevent inactivation of the antigen in a solution containing the antigen and can be stably stored.

EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to this.
<Example 1>
a) Preparation of antigen-containing aqueous solution A 100 mM ADA buffer (pH 6.6) was prepared by a conventional method. Next, the respective concentrations were added to the weight of the antigen-containing aqueous solution such that casein (milk) 0.5% by weight and whey protein (milk) 0.5% by weight, and dissolved by heating.
After confirming uniform dissolution, an N-terminal brain natriuretic peptide precursor was added to a concentration of 5000 pg / g to prepare an antigen-containing aqueous solution 1.
b) Evaluation of Storage Stability The prepared antigen-containing aqueous solution 1 is stored at 4 ° C. and 25 ° C., and at the time of preparation, the N-terminal brain natriuretic peptide precursor concentration at the 3rd, 6th and 12th months is expressed as N Measurement was performed with a terminal brain natriuretic peptide precursor measurement reagent {manufactured by Sanyo Kasei Kogyo Co., Ltd.} to examine the change in concentration. The results are shown in Table 2.

<Examples 2-9 and Comparative Examples 1-3>
In Example 1, the antigen-containing aqueous solutions 2 to 9 were used in the same manner as in Example 1 except that the buffer type, pH, casein, whey protein and N-terminal brain natriuretic peptide precursor concentrations were changed to the values shown in Table 1. And comparative antigen-containing aqueous solutions 1 to 3 were obtained. These were evaluated in the same manner as in Example 1, and the results are shown in Table 2.

  In the antigen-containing aqueous solution of the present invention, the concentration change of the N-terminal brain natriuretic peptide precursor was small compared to the comparative antigen-containing aqueous solution. In particular, Example 1, Example 4, Example 5, Example 6, and Example 7 were very stable even after 12 months.

The antigen-containing aqueous solution of the present invention has the following effects.
(1) The N-terminal brain natriuretic peptide precursor which is easily inactivated can be stably stored for a long time in a solution state.
(2) The stability can be maintained for a long time without lyophilization performed in the past, and there is no concentration error caused by a complicated preparation operation of dissolving the antigen, and immunoassay with good measurement accuracy and reproducibility is achieved. it can.
Therefore, the antigen-containing aqueous solution of the present invention can be used when performing immunoassay such as enzyme immunoassay, radioimmunoassay, or immunoturbidimetry in the field of clinical examination. In particular, it can be suitably used as a standard solution because it can prevent inactivation of the antigen in the solution containing the antigen and can be stably stored.

Claims (2)

An N-terminal brain natriuretic peptide precursor, casein and / or whey protein, and a buffer containing at least one functional group selected from the group consisting of an amide group, a sulfo group and a carboxyl group, An antigen-containing aqueous solution for a standard solution used for measuring the content of an N-terminal brain natriuretic peptide precursor by an immunoassay characterized by a pH of 5.5 to 7.0. The antigen-containing aqueous solution according to claim 1, wherein the casein and / or whey protein concentration is 0.01 to 3% based on the weight of the antigen-containing aqueous solution.