JP2012139167A - Stabilized s-adenosylhomocysteine hydrolase preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To stably maintain an S-adenosylhomocysteine hydrolase to be used in an immunoassay measuring method of a homocysteine, in a freeze-dried condition or the like.SOLUTION: Coexisting of the S-adenosylhomocysteine hydrolase and a collagen peptide stably maintains the S-adenosylhomocysteine hydrolase in a solution condition, in a frozen condition, or in the freeze-dried condition, and prevents its deactivation.

Description

  The present invention relates to a preparation of stabilized S-adenosylhomocysteine hydrolase. The preparation of the present invention is provided in the form of an aqueous solution, frozen state or lyophilized state. For example, homocysteine contained in blood is converted to S-adenosylhomocysteine and then bound to a labeling substance. The present invention provides a stable preparation in which the inactivation of S-adenosylhomocysteine hydrolase, which is necessary when immunoassay is performed using an anti-S-adenosylhomocysteine antibody, is prevented. is there.

  Homocysteine is an intermediate metabolite of methionine metabolism that is an essential amino acid of the SH-containing group. The high homocysteine level is caused by a genetic metabolic disorder known as homocysteineuria, and also due to nutritional disorders (folic acid, vitamin B6, vitamin B12 deficiency) and the like. In recent years, attention has been paid to the relationship between homocysteine and vascular disorders, and the relationship between homocysteineuria and arteriosclerosis has been reported, and it has become clear that it is an independent risk factor for thrombosis, cardiovascular disease, and cerebral infarction. Yes. (Non-Patent Documents 1 and 2). Based on such findings, measurement of the total homocysteine amount present in blood is performed using urine or blood as a sample.

  Known methods for measuring homocysteine include a method using liquid chromatography and an immunoassay method. Among them, the immunoassay method is frequently used in the measurement of the amount of homocysteine performed for clinical purposes because of its rapidity.

  As immunoassay, homocysteine in a sample is reduced to a free state and further converted to S-adenosylhomocysteine, and an antibody against S-adenosylhomocysteine (anti-S-adenosine). A method for measuring S-adenosylhomocysteine by a so-called competition method using a sylhomocysteine antibody) is known. In this pretreatment step, homocysteine (Non-patent Document 3) present as an oxidized disulfide compound in the sample is treated with a reducing agent, and the conversion to S-adenosylhomocysteine is performed by adenosine. In the presence of S-adenosylhomocysteine hydrolase.

McCully, K.M. S. Carvalho, A .; V. A. : Homocysteine thiolactone, n-homocysteine thiolactonyl retinamide, and platelet aggregation. Res. Commum. Chem. Pathol. Pharmacol. 56: 349-360, 1987. Sasaki, et al. : Relationship between arteriosclerotic disease and blood homocysteine level. Progress in Medicine. 21, 6: 1543-1549, 2001. D. W. Jacobsen Homocystein and vitamins in cardiovascular disease. Clin. Chem. , August 1, 1998; 44 (8): 1833-1843.

  As described above, S-adenosylhomocysteine hydrolase is used in the pretreatment step for measuring the total amount of homocysteine. This enzyme has poor stability in a solution state, and even when a reagent for stabilizing a protein such as a reducing agent, EDTA, glycerol solution and / or bovine serum albumin (BSA) is generally present at a low temperature. Need to save. Therefore, there is a problem that handling is difficult, for example, deterioration proceeds only at room temperature in order to measure homocysteine.

  Accordingly, an object of the present invention is to provide a preparation of S-adenosylhomocysteine hydrolase that is stable even at room temperature.

  The present inventors have intensively studied to achieve the above object, and completed the present invention. The present invention that has achieved the above object is a stabilized S-adenosylhomocysteine hydrolase preparation comprising S-adenosylhomocysteine hydrolase and a collagen peptide. Hereinafter, the present invention will be described in detail.

  The present invention is a preparation of S-adenosylhomocysteine hydrolase. The preparation of the present invention includes a solution containing S-adenosylhomocysteine hydrolase, a frozen product of the solution, and the frozen product. A dried lyophilized product is included. Among them, the effect of the present invention is remarkable for freeze-dried products. The S-adenosylhomocysteine hydrolase contained in the preparation is collected from human placenta or a gene encoding S-adenosylhomocysteine hydrolase (cDNA, S-adenosylhomocysteine hydrolase). The nucleotide sequence of the gene coding for is known to have been genetically engineered by incorporating it into other microorganisms, or the amino acid primary structure of a naturally occurring S-adenosylhomocysteine hydrolase Is a mutant S-adenosylhomocysteine hydrolase-related enzyme into which is introduced. There is no particular limitation on the amount (concentration) of S-adenosylhomocysteine hydrolase contained in the preparation. The preparation of the present invention is a container made of various materials such as glass and plastic, and can be placed in containers of various shapes such as vials, plates and cups.

  The present invention is characterized in that a collagen peptide coexists with S-adenosylhomocysteine hydrolase. There is no particular limitation on the origin of the collagen peptide as long as it is obtained by hydrolyzing collagen. For example, collagen derived from pig bone, pig skin, fish scale, cow bone, cow skin, etc. is hydrolyzed. Degraded collagen peptides can be exemplified. Among them, those derived from pig skin are particularly preferable from the viewpoint that they are abundant in raw materials and are easily available. The degree of hydrolysis is not particularly limited. For example, collagen may be heated, for example, at 50 ° C. for 180 minutes in the presence of a protein hydrolase such as papain, pepsin or trypsin.

  The amount of collagen peptide used, that is, the amount (concentration) to coexist with S-adenosylhomocysteine hydrolase is 1 to 10% by weight, more preferably 1 to 5% by weight in the preparation in the solution state. it can. In producing the preparation of the present invention, S-adenosylhomocysteine hydrolase may be added to a solution containing collagen peptide, or conversely, a solution containing S-adenosylhomocysteine hydrolase. Collagen peptide may be added to. In order to produce a frozen product or a lyophilized product, the solution thus produced may be simply frozen and then dried.

  The preparation of the present invention contains S-adenosylhomocysteine hydrolase and collagen peptide, but further enhances the stabilizing effect of S-adenosylhomocysteine hydrolase by collagen peptide by coexisting with trehalose. be able to. There is no restriction | limiting in particular as a trehalose, For example, using a commercially available trehalose can be illustrated. When trehalose is further coexisted, the amount (concentration) to be used is 1 to 10% by weight, preferably 2 to 5% by weight, in the preparation in the solution state.

  As described above, the preparation of the present invention contains S-adenosylhomocysteine hydrolase and collagen peptide, and preferably further contains trehalose, but these preparation components are added to a buffer containing an appropriate buffer. It is preferably in a dissolved solution state, or after being dissolved in an appropriate buffer solution, it is then frozen and lyophilized. Examples of the buffer solution include a GOOD buffer solution such as MES, Bis-Tris, ADA, PIPES, ACES, BES, MOPS, TES, HEPPES, Tricine, Bicine, CHES, and CAPS, as well as Tris buffer and phosphate buffer. However, it is preferable to maintain the pH at a pH of 5.0 to 9.0, preferably 7.0 to 7.5, in which S-adenosylhomocysteine hydrolase is stable, in a solution state with such a buffer. .

  The preparation of the present invention can further contain components other than the buffer as long as the stability of the S-adenosylhomocysteine hydrolase is not affected. Such components include, for example, preservatives that prevent the growth of microorganisms, surfactants for dispersing and solubilizing fat-soluble components, antioxidants for preventing oxidation, and bonds for maintaining the shape of the preparation. Agent, swelling agent for protecting S-adenosylhomocysteine hydrolase, colorant for distinguishing the preparation from other reagents, etc., for maintaining the structure of S-adenosylhomocysteine hydrolase Suspending agents, emulsifiers for dispersing and solubilizing the components in the preparation, solubilizing agents for homogenizing the preparation solution, maintaining the structure of S-adenosylhomocysteine hydrolase, etc. A tonicity agent etc. can be illustrated. Examples of antiseptics include sodium azide, chelating agents, antibiotics, antibacterial agents, and antifungal agents. Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants. Examples of the antioxidant include ascorbic acid and vitamin E. Examples of the binder include carboxymethyl cellulose. Examples of the wetting agent include cellulose and polyethylene glycol. Examples of the colorant include synthetic food colorants. Examples of the suspending agent include polyvinyl pyrrolidone. Examples of the emulsifier include alkyl sulfonic acid. Examples of solubilizers include glycerin. Examples of the isotonic agent include D-sorbitol and sodium chloride. These components can be included in the preparation at any time according to the purpose of use, but the amount (concentration) is subjected to a preliminary test in which various concentrations coexist. What is necessary is just to determine the density | concentration which can achieve a desired effect.

  The preparation of the present invention is used, for example, in a pretreatment step when performing immunoassay. In the pretreatment step in this immunoassay, the homocysteine in the sample is reduced, and the reduced homocysteine is contacted with S-adenosylhomocysteine hydrolase in the presence of adenosine to convert to S-adenosylhomocysteine. It is a process. When used in this step, the preparation of the present invention and the reducing agent are not mixed until immediately before the pretreatment step, but are kept separated so that S-adenosylhomocysteine hydrolase is stable. It is preferable to hold it. Specifically, first, one or more reducing agents selected from dithiostatol (DTT), tris (carboxyethyl) phosphine (TCEP), mercaptoethanol and the like are added to the sample to reduce homocysteine, and then the present invention. The above preparation and adenosine may be added to the sample, or the reducing agent and the preparation of the present invention and adenosine may be added to the sample simultaneously. The amount of adenosine to be added may be appropriately determined within the range in which the reverse reaction does not occur, but it is usually sufficient to be within the range of 0.04 to 0.2 mol / L.

The S-adenosylhomocysteine hydrolase preparation of the present invention is preserved while maintaining the activity of S-adenosylhomocysteine hydrolase stably in any state of solution, frozen state or lyophilized state. Is possible. Moreover, since the preparation of the present invention can be produced by a very simple operation of adding a collagen peptide to a solution containing S-adenosylhomocysteine hydrolase, the time required for producing the reagent is short, In addition, it does not require a large cost for manufacturing.
When the preparation of the present invention is used, for example, as an immunoassay reagent that requires conversion of homocysteine in a sample to S-adenosylhomocysteine, it can be stored for a long time while retaining its activity. With this advantageous effect of the present invention, it is possible to provide a measurement reagent that does not deteriorate in quality over a longer period of time. Furthermore, according to the preparation of the present invention that can stably maintain the activity, it is possible to improve reproducibility between measurements in immunoassays.

EXAMPLES Hereinafter, although an Example demonstrates embodiment of this invention, this invention is not limited to these Examples.

Example 1 Production of S-adenosylhomocysteine hydrolase preparation 1
In a glass vial, swine was adjusted to 5.0% by weight with respect to 100 mmol / L Tris buffer (pH 8.0) containing BSA and 40 kilounit / L S-adenosylhomocysteine hydrolase. A skin-derived collagen peptide was added, and trehalose was further added to a concentration of 5.0% by weight to produce the preparation of the present invention. In addition, for comparison, one without collagen peptide and / or one without trehalose is prepared, and after freezing, further dried, enzyme activity immediately after freeze-drying, after freeze-drying, at 40 ° C. for 7 days The enzyme activities of the preparations after storage were measured and compared.

  The enzyme activity was measured using a commercially available immunoassay device (trade name: AIA-2000, manufactured by Tosoh Corporation), and after subjecting the blood sample to a pretreatment step, was performed by a one-step competition method.

  As a reagent for immunoassay, (a) a conjugate of an anti-S-adenosyl-L-homocysteine antibody prepared using a mouse and alkaline phosphatase (ALP) as a labeling substance, and (a) an artificially prepared S -A conjugate of adenosyl-L-homocysteine and BSA, which further binds the BSA fluorescein, and (c) an anti-fluorescein antibody prepared using a mouse, having a diameter of about 2 mm A reagent containing a spherical solid phase was used. This reagent is prepared by dispensing (a) and (c) in a plastic cup and freezing, further dispensing (a) and freezing, and then lyophilizing (a) to (c). It is a reagent manufactured in this way.

  In the pretreatment, as a pretreatment reagent (1) containing a reducing agent and adenosine, 10 mmol / L Bis-Tris buffer containing 4.0 mmol / L TCEP and 8.0 mmol / L adenosine is used. This was mixed with a preparation containing S-adenosylhomocysteine hydrolase prepared as described above, then mixed with a blood-derived sample, and stirred at 37 ° C. for 10 minutes. Then, B / F separation operation was implemented by the B / F separation mechanism of the apparatus, and the amount of the labeling substance (ALP) contained in the component remaining in the cup as a solid phase component was measured. ALP was measured by adding 4-methylumbelliferyl phosphate (4MUP), its substrate, and increasing rate (nmol / sec) of 4-methylumbelliferone (4MU) produced by decomposition of 4MUP by ALP. The fluorescence intensity from 4MU was measured and calculated.

  The results are shown in Table 1. In Table 1, the HPLC measurement value indicates the amount (μmol / L) of S-adenosylhomocysteine hydrolase in each preparation determined by the HPLC method, and each measurement value is a measurement result. The amount of S-adenosylhomocysteine hydrolase calculated from (μmol / L) is shown. Moreover, enzyme activity (%) shows the average value of the ratio of the quantity of S-adenosyl homocysteine hydrolase measured by HPLC method, and the quantity of S-adenosyl homocysteine hydrolase computed from the measurement result. Is. From Table 1, it can be seen that the S-adenosylhomocysteine hydrolase in the preparation can be stably retained when the collagen peptide and trehalose coexist.

Example 2 Production of S-adenosylhomocysteine hydrolase preparation 2
In a glass vial, swine was adjusted to 5.0% by weight with respect to 100 mmol / L Tris buffer (pH 8.0) containing BSA and 40 kilounit / L S-adenosylhomocysteine hydrolase. A skin-derived collagen peptide was added, and trehalose was further added to a concentration of 5.0% by weight to produce the preparation of the present invention. For comparison, a comparative preparation was prepared by adding sucrose to 5.0% by weight in place of trehalose. After freezing, these were further dried, and enzyme activity immediately after lyophilization, after lyophilization, 40 The enzyme activity of the preparations after storage at 7 ° C. for 7 days was measured and compared.

  The enzyme activity was measured using a commercially available immunoassay device (trade name: AIA-2000, manufactured by Tosoh Corporation), and after subjecting the blood sample to a pretreatment step, was performed by a one-step competition method.

  As a reagent for immunoassay, (a) a conjugate of an anti-S-adenosyl-L-homocysteine antibody prepared using a mouse and alkaline phosphatase (ALP) as a labeling substance, and (a) an artificially prepared S -A conjugate of adenosyl-L-homocysteine and BSA, which further binds the BSA fluorescein, and (c) an anti-fluorescein antibody prepared using a mouse, having a diameter of about 2 mm A reagent containing a spherical solid phase was used. This reagent is prepared by dispensing (a) and (c) in a plastic cup and freezing, further dispensing (a) and freezing, and then lyophilizing (a) to (c). It is a reagent manufactured in this way.

  In the pretreatment, as a pretreatment reagent (1) containing a reducing agent and adenosine, 10 mmol / L Bis-Tris buffer containing 4.0 mmol / L TCEP and 8.0 mmol / L adenosine is used. This was mixed with the S-adenosyl homocysteine hydrolase preparation prepared as described above, then mixed with the blood-derived sample, and stirred at 37 ° C. for 10 minutes. Then, B / F separation operation was implemented by the B / F separation mechanism of the apparatus, and the amount of the labeling substance (ALP) contained in the component remaining in the cup as a solid phase component was measured. ALP was measured by adding 4-methylumbelliferyl phosphate (4MUP), its substrate, and increasing rate (nmol / sec) of 4-methylumbelliferone (4MU) produced by decomposition of 4MUP by ALP. The fluorescence intensity from 4MU was measured and calculated.

  The results are shown in Table 2. In Table 2, each measured value indicates the activity of the S-adenosylhomocysteine hydrolase calculated from the measurement results, and the activity immediately after preparing the preparation is defined as 100%. Activity after storage at 7 ° C. for 7 days is shown. From Table 2, it can be seen that the combination of collagen peptide and trehalose is the most effective for stably storing S-adenosylhomocysteine hydrolase.

Example 3 Immunoassay of homocysteine in a sample Using a commercially available immunoassay device (trade name AIA-2000, manufactured by Tosoh Corporation), a blood sample was subjected to a pretreatment step, and then subjected to S- Adenosyl homocysteine (homocysteine) was measured.

  A 10 mmol / L Bis-Tris buffer solution containing 4.0 mmol / L TCEP and 8.0 mmol / L adenosine was prepared as a pretreatment reagent (1) containing a reducing agent and adenosine. As a pretreatment reagent (2) containing S-adenosylhomocysteine hydrolase, a preparation containing porcine skin-derived collagen peptide and trehalose among the preparations produced in Example 2 was used.

  As a reagent for immunoassay, (a) a conjugate of an anti-S-adenosyl-L-homocysteine antibody prepared using a mouse and alkaline phosphatase (ALP) as a labeling substance, and (a) an artificially prepared S -A conjugate of adenosyl-L-homocysteine and BSA, which further binds the BSA fluorescein, and (c) an anti-fluorescein antibody prepared using a mouse, having a diameter of about 2 mm A reagent containing a spherical solid phase was used. This reagent is prepared by dispensing (a) and (c) in a plastic cup and freezing, further dispensing (a) and freezing, and then lyophilizing (a) to (c). It is a reagent manufactured in this way.

  The measurement was performed with the above-described apparatus. The mixed solution of the pretreatment reagent 1 and the pretreatment reagent 2 was first mixed with the blood-derived sample and stirred at 37 ° C. for 10 minutes. Then, B / F separation operation was implemented by the B / F separation mechanism of the apparatus, and the amount of the labeling substance (ALP) contained in the component remaining in the cup as a solid phase component was measured. ALP was measured by adding 4-methylumbelliferyl phosphate (4MUP), its substrate, and increasing rate (nmol / sec) of 4-methylumbelliferone (4MU) produced by decomposition of 4MUP by ALP. The fluorescence intensity from 4MU was measured and calculated.

  As a result, the preparation of the present invention in which pig skin collagen peptide and trehalose were added and lyophilized had suppressed inactivation of S-adenosylhomocysteine hydrolase, and the pretreatment reagent as described above As a result, it was possible to obtain good immunoassay results.

Claims (3)

  A stabilized S-adenosylhomocysteine hydrolase preparation comprising S-adenosylhomocysteine hydrolase and a collagen peptide.   The stabilized S-adenosylhomocysteine hydrolase preparation of claim 1, further comprising trehalose.   3. Stabilized S-adenosylhomocysteine hydrolase preparation according to claim 1 or 2, characterized in that it is freeze-dried.