JP2012141171A - Cardiac disease marker standard sample and method of manufacturing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a standard sample which simultaneously and stably contains cTn and BNP which are especially important as a cardiac marker and can be used as a control material for measurement in an immunoassay.SOLUTION: This invention provides cardiac troponin and brain natriuretic peptide-containing standard samples characterized by coexistence of a casein-based protein and a benzamidine derivative.

Description

  The present invention relates to a preparation containing myocardial troponin (Cardiac Troponin, hereinafter referred to as cTn) and brain natriuretic peptide (hereinafter referred to as BNP) and a method for producing the same. The preparation of the present invention is useful as a management substance for measurement when cTn and / or BNP are measured as a heart disease marker in immunoassay, for example.

  Various heart disease markers such as myoglobin, creatine kinase MB (CKMB), cTn (having three subunits cTnI, cTnT and cTnC derived from the heart) or BNP have become known, and are widely used in the field of clinical examination. It has been measured (Non-Patent Document 1).

  Among various myocardial markers, cTn is the latest guideline (Japanese cardiovascular society acute myocardial infarction (ST elevation type) clinical guidelines, guidelines for diagnosis and treatment of acute and chronic myocarditis, European Heart Society / American Heart Disease) It is used as an index of myocardial injury in acute coronary syndrome (ACS) in the guidelines for diagnostic criteria of myocardial infarction of the academic society. BNP is a sensitive biomarker of heart failure and is widely used not only for diagnosis of heart failure but also for prognosis evaluation and treatment determination. For this reason, measurement of cTn and BNP among heart disease markers is particularly important in the diagnosis of heart diseases.

  As a method for measuring cTn and BNP, immunoassay is widely used. When measuring cTn and BNP by immunoassay, the same result will be obtained if cTn, etc. of the same concentration exists even if the person who performs the measurement, the apparatus that performs the measurement, and the date and time of the measurement are different. Accuracy control is important. For this reason, a measurement control substance, which is a preparation containing a known concentration of cTn or a known concentration of BNP, has been used. However, cTn or BNP contained therein is stable in the measurement control substance. It is required that there be little change in the measured value over time when cTn or BNP immunoassay is performed on the measurement control substance, and that it does not interfere with the reaction with the antigen or antibody in the immunoassay.

  Since the importance of cTn and BNP is increasing among various cardiac markers, a substance for measurement management containing both cTn and BNP should be provided to improve convenience. There is a difference between the conditions under which cTn can be stably present and the conditions under which BNP can be stably reported (Patent Document 1), and the measurement includes both cTn and BNP. No controlled substances have been reported yet. In particular, BNP easily adsorbs glass, and when stored in a glass container, there is a problem that the above-described change in measured value over time is larger than when stored in a polystyrene or polypropylene container.

JP2010-091398A

THROMBOSIS and Circulation Vol. 13 No. 2 2005 81 (189-193)

  Accordingly, an object of the present invention is to provide a preparation that contains cTn and BNP, which are particularly important as myocardial markers, at the same time and stably, and can also be used as a measurement management substance in immunoassay.

  In order to achieve the above object, the present inventors have intensively studied, and as a result, the present invention has been completed. The present invention that achieves the above object is a cTn- and BNP-containing preparation characterized by coexisting a casein protein and a benzamidine derivative. Moreover, this invention which achieves the said objective is a manufacturing method of a cTn and a BNP containing sample characterized by adding casein type protein and a benzamidine derivative. Hereinafter, the present invention will be described in detail.

  The present invention is a standard containing both cTn and BNP. The preparations used in the present invention include measurement control substances used in enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), etc. In addition to those containing cTn and BNP at strictly controlled concentrations, which are also used as standard products for preparing calibration curves, they may simply contain cTn or BNP. Examples of substances used as measurement control substances include those in which cTn is controlled in the range of 0.2 to 50 ng / mL and BNP is controlled in the range of 20 to 2000 pg / mL. The preparation may be in the form of a solution, frozen, or dried after freezing. In particular, the present invention is particularly preferably applied to a measurement-controlled substance in a lyophilized state that is provided in a lyophilized state and dissolved upon use.

  The cTn contained in the preparation is one or more selected from cTnI, cTnT, cTnC, cTnI and T dimer, cTnI and C dimer, cTnT and C dimer, or cTcI, T and C trimer. If it exists, the stabilization effect by coexistence of a casein protein and a benzamidine derivative can be obtained in any form. Both cTn and BNP contained in the preparation are not limited to those derived from humans, and may be, for example, cTn or BNP derived from rats, pigs, dogs or eels. In addition to products, chemical synthesis products, products produced by gene recombination techniques, and the like can also have a stabilizing effect due to the coexistence of casein proteins and benzamidine derivatives. Furthermore, for example, in BNP, a derivative having a ring site formed by, for example, a -SS-bond may be used. As an example of the BNP derivative in the present invention, so-called Pro BNP and NT-Pro BNP can be exemplified. Furthermore, for example, ANP, CNP and the like in which the content ratio of each amino acid residue in the primary structure is similar can also be exemplified as the BNP derivative in the present invention.

  As the casein protein, various caseins such as plant-derived casein and milk casein and sodium caseinate can be used. As a commercially available product, for example, a product distributed as a solid phase blocking agent for immunoassay (trade name Block Ace, manufactured by DS Pharma Biomedical Co., Ltd.) may be used. The casein protein may be determined in consideration of the coexisting amount of cTn and BNP in the preparation. For example, when the preparation of the present invention is used as a controlled substance for measurement, the cTn is generally 0.2 to 50 ng. / ML and BNP are in the range of 20 to 2000 pg / mL, so that in the solution state, the final concentration is preferably 1 to 10%, particularly preferably 1 to 3%.

  Examples of benzamidine derivatives include benzamidine (hydrochloride), aminobenzamidine, 4-methoxybenzamidine and the like. The amount of coexistence of the benzamidine derivative may be determined in consideration of the cTn and BNP concentrations in the sample. For example, when the sample of the present invention is used as a measurement control substance, generally the cTn is 0.2 to 50 ng / Since mL and BNP are in the range of 20 to 2000 pg / mL, it can be exemplified that in a solution state, the final concentration is preferably 1 to 100 mM, particularly preferably 5 to 20 mM.

  In addition to casein proteins and benzamidine derivatives, the preparation of the present invention, depending on the purpose, for example, a preservative such as bovine serum albumin, an antioxidant such as ascorbic acid and vitamin E, a binder such as carboxymethyl cellulose, For example, a wetting agent such as cellulose or polyethylene glycol, a coloring agent such as a synthetic food colorant, a suspending agent such as polyvinylpyrrolidone, an emulsifier such as an alkyl sulfonic acid, a solubilizing agent such as glycerin, such as a phosphate or the like Buffers such as trishydroxylamine hydrochloride, for example, isotonic agents such as D-sorbitol or sodium chloride, for example, surfactants such as Triton X-100 or Tween 20 may be included. When one or more of the agents exemplified above are further allowed to coexist, for example, if the sample is used as a measurement control substance, it is confirmed that the antigen-antibody reaction is not inhibited. It is preferable to consider the use of the product. Furthermore, in the present invention, myoglobin and CKMB, which are myocardial markers other than cTn or BNP, can be contained as long as their stability can be ensured.

  In the present invention, the preparation is produced by, for example, mixing a solution containing cTn and BNP with a solution containing a casein protein and a benzamidine derivative. In this operation, in order to prevent adsorption of BNP to glass, if a container made of polystyrene or polypropylene is used, or if a glass container is used, first, a solution containing casein protein and benzamidine derivative is added to glass. It is preferable to put in a container and then add a solution containing cTn and BNP. In addition, by coexisting a casein protein and a benzamidine derivative, it is possible to reduce adsorption of BNP to glass.

  The preparation of the present invention produced as described above will not lose its stability even if it is then frozen and further dried if necessary.

  According to the present invention, a preparation containing both cTn and BNP, which is increasing in importance in the field of diagnosis, can be stabilized over a long period of time by an extremely simple operation of coexisting a casein protein and a benzamidine derivative. Can be maintained. Moreover, since the effect of coexisting a casein protein and a benzamidine derivative is not lost even in a frozen state or a lyophilized state as well as a liquid state, it is possible to provide preparations in various forms. . Furthermore, the adsorption of BNP to the glass is suppressed, and it becomes possible to use a glass container that has been difficult to use for storing BNP conventionally.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples. In each case, the casein protein used was commercially available as a solid phase blocking agent for immunoassay (trade name Block Ace, manufactured by DS Pharma Biomedical Co., Ltd.).

Example 1
BNP, cTnI, myoglobin in 20 mM Tris-HCl buffer (pH 7.5) containing 2 wt% casein protein so that the final concentrations are 40 pg / mL, 0.5 ng / mL, 50 ng / mL, or 10 ng / mL, respectively. And CKMB were added to prepare Solution A. Benzamidine hydrochloride was added to Solution A so as to have a final concentration of 5 mM or 10 mM, respectively, to prepare Solution B and Solution C.

  2 mL of each of solutions A to C was dispensed into glass containers and freeze-dried, and then stored at 40 ° C. for 7 days. Thereafter, 2 mL of distilled water was added to the lyophilized product and dissolved, and BNP, troponin I, myoglobin, and CKMB concentrations were measured. The results are shown in Table 1. In Table 1, “concentration percentage” is calculated based on Equation 1 below.

  Table 1 shows that the concentration percentages of BNP, cTnI, myoglobin, and CKMB are 90% or more in solution B and solution C (samples in which casein protein and benzamidine hydrochloride coexist) even after storage at 4 ° C. for 7 days. It can be seen that these myocardial markers are stable.

Comparative Example 1
BNP, cTnI, myoglobin, and CKMB in 20 mM Tris-HCl buffer (pH 7.5) containing 5 wt% bovine serum albumin so that the final concentrations are 100 pg / mL, 5 ng / mL, 100 ng / mL, or 100 ng / mL, respectively. Was added to prepare Solution D. Solution E was prepared in the same manner for 20 mM Tris-HCl buffer (pH 7.5) containing gericate peptone, and Solution F was prepared in the same manner for 20 mM Tris-HCl buffer (pH 7.5) containing casein protein. did.

After 2 mL of each of solutions D to F was dispensed into a glass container and freeze-dried, 4 mL
Stored at 7 ° C for 7 days. Thereafter, 2 mL of distilled water was added to the lyophilized product and dissolved, and BNP, troponin I, myoglobin, and CKMB concentrations were measured. The results are shown in Table 2. In Table 2, “concentration percentage” is calculated based on the following formula 2.

  Table 2 shows that in solution F (a sample in which casein protein coexists), BNP, troponin I, myoglobin, and CKMB are stored at 40 ° C. for 7 days, and the concentration percentage is 90% or more, which is relatively good. However, especially for BNP, it can be seen that the result in Example 1 is lower.

  From the above results, it can be seen that BNP, troponin I, myoglobin, and CKMB cannot be stably stored in a preparation in which neither casein protein nor benzamidine derivative coexists. In addition, although a certain effect can be obtained with a casein protein alone, it can be seen that the effect of improving the stability does not reach the effect of coexisting a casein protein and a benzamidine derivative.

Claims (2)

  A preparation containing cardiac troponin and brain natriuretic peptide, characterized by coexisting a casein protein and a benzamidine derivative.   A method for producing a preparation containing cardiac troponin and brain natriuretic peptide, comprising adding a casein protein and a benzamidine derivative.