Leucine-rich alpha 2 glycoprotein composition

Abstract

An object of the present invention is to provide a leucine-rich α2 glycoprotein composition, which comprises leucine-rich α2 glycoprotein and has excellent storage stability. The object of the present invention can be achieved by a leucine-rich α2 glycoprotein composition comprising leucine-rich α2 glycoprotein and having a pH of 7.0 to 9.3.

Description

Technical Field

[0001] The present invention relates to a composition of α2 glycoprotein rich in leucine, and more particularly to a composition of α2 glycoprotein rich in leucine that can stably preserve the composition for a long period of time. Background Art

[0002] Leucine-rich α2 glycoprotein (hereinafter sometimes referred to as LRG) is one of the serum proteins. It is reported that LRG is a glycoprotein of about 50 kDa secreted by neutrophils (Non-patent Document 1).

[0003] Recently, the correlation between LRG in biological samples and specific diseases has been studied. For example, Patent Document 1 reports that detecting LRG in biological samples is useful for detecting autoimmune diseases such as Behcet's disease.

[0004] In order to quantitatively measure the component to be measured in a biological sample, a calibration sample must be used. The calibration sample is a sample containing the component to be measured and is used as an internal standard, a concentration calibration standard (calibrator), etc. In order to obtain an accurate quantitative value, a calibration sample that is stable against the passage of time or temperature is required. That is, it is believed that when the component to be measured in the calibration sample is decomposed and / or denatured during storage, the measured value is also affected. For the quantitative measurement of LRG, methods using enzyme-linked immunosorbent assay (ELISA) and the like have been known so far, but calibration samples have not been examined.

[0005] Citation List

[0006] Patent Literature

[0007] Patent Document 1: JP-A-2010-286279

[0008] Non-patent literature

[0009] Non-patent document 1: J Leukoc Biol. 2002 72(3): 478-85.2002 Summary of the invention

[0010] Technical issues

[0011] The present inventors attempted to produce a calibration sample solution containing LRG. As a result, it was found that when LRG was added to a solution with a pH of less than 7, the stability of LRG was poor, and LRG was gradually decomposed and lost over time.

[0012] An object of the present invention is to provide a leucine-rich α2 glycoprotein composition, which comprises leucine-rich α2 glycoprotein and has high storage stability.

[0013] Solutions to the problem

[0014] As a result of further studies, the inventors found that when the pH of a solution containing LRG is maintained within a specific range, the stability of LRG is improved. It was also found that when the salt concentration of the solution is within a specific range, the stability of LRG is further improved. The present invention is based on these findings.

[0015] Specifically, the present invention is as follows:

[0016] <1> A leucine-rich α2 glycoprotein composition comprising leucine-rich α2 glycoprotein, wherein the pH of the composition is 7.0 to 9.3,

[0017] <2> according to <1> A leucine-rich α2-glycoprotein composition, which is a calibration sample solution for measuring leucine-rich α2-glycoprotein,

[0018] <3> according to <1> or <2> A leucine-rich α2 glycoprotein composition filled in a storage container,

[0019] <4> according to <1> to <3> The leucine-rich α2 glycoprotein composition of any one of claims , wherein the pH of the composition is 7.8 to 9.0,

[0020] <5> according to <1> to <4> The leucine-rich α2 glycoprotein composition of any one of the above, wherein the salt concentration of the composition is 200 mM to 800 mM,

[0021] <6> according to <5> The leucine-rich α2 glycoprotein composition of claim 1, wherein the salt is a metal salt,

[0022] <7> A method for measuring leucine-rich α2 glycoprotein, comprising:

[0023] Use according to <1> to <6> The leucine-rich α2 glycoprotein composition of any one of the above,

[0024] <8> A kit for determining leucine-rich α2 glycoprotein, comprising the method according to claim <1> to <6> The leucine-rich α2 glycoprotein composition of any one of the above,

[0025] <9> A method for stabilizing the storage of leucine-rich α2 glycoprotein, comprising:

[0026] contacting the leucine-rich alpha 2 glycoprotein with a solvent having a pH value of 7.0 to 9.3,

[0027] <10> according to <9> A method for stabilizing the storage of leucine-rich α2 glycoprotein, wherein the pH of the solvent is 7.8 to 9.0,

[0028] <11> according to <9> or <10> A method for stabilizing the storage of leucine-rich α2 glycoprotein, wherein the salt concentration in the solvent is 200 mM to 800 mM, and

[0029] <12> according to <9> to <11> The method for storage stabilization of leucine-rich α2-glycoprotein according to any one of the preceding claims, wherein the salt is a metal salt.

[0030] Advantageous Effects of the Invention

[0031] According to the present invention, a composition containing LRG that contains leucine-rich α2-glycoprotein and has excellent storage stability can be provided, and in particular, a calibration sample containing LRG can be provided. Therefore, according to the present invention, LRG in a biological sample can be accurately quantitatively measured, and a disease can be accurately diagnosed. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] [ Figure 1 ] shows a graph showing the change in the amount of LRG of solutions with different pH values ​​over time, where the initial value is considered to be 100%.

[0033] [ Figure 2 ] shows a graph of the change in the amount of LRG of solutions with different salt concentrations over time, where the initial value is considered to be 100%.

[0034] [ Figure 3 ] shows a graph showing the change in the amount of LRG of solutions with different pH values ​​over time, where the initial value is considered to be 100%. DETAILED DESCRIPTION

[0035] (Leucine-rich alpha 2 glycoprotein)

[0036] Leucine-rich α2 glycoprotein or LRG is a glycoprotein of about 50 kDa. LRG is one of the serum proteins and is said to contain about 3.0 μg / mL in the serum of healthy individuals. LRG is reported to be secreted by neutrophils. In recent years, the correlation between changes in the amount of LRG in the body and various diseases such as tuberculosis and tumors has been studied, and in order to accurately diagnose these diseases, it is necessary to accurately measure the amount of LRG in the body.

[0037] LRG can be measured using known methods such as immunological methods. Immunological methods include ELISA, enzyme immunoassay, surface plasmon resonance, latex agglutination immunoassay (LTIA), chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescent antibody technology, radioimmunoassay, immunoprecipitation, Western blotting, immunochromatography, high performance liquid chromatography (HPLC), etc.

[0038] As the LRG contained in the composition of the present invention, commercially available LRG may be used, or LRG produced or purified by the user may be used. As the LRG contained in the composition of the present invention, LRG produced in vitro may be used, or LRG extracted from a living body may be used.

[0039] (pH)

[0040] Considering the stability of LRG, the pH of the leucine-rich α2 glycoprotein composition of the present invention is 7.0 to 9.3, preferably 7.0 to 9.0, more preferably 7.5 to 9.0, further preferably 7.8 to 9.0, and most preferably 8.0 to 9.0.

[0041] The pH can be adjusted using pH adjusting agents known to those skilled in the art, such as sodium hydroxide.

[0042] (Salt concentration)

[0043] The salt concentration of the LRG composition of the present invention is preferably 200 mM to 800 mM, more preferably 300 mM to 700 mM, and most preferably 300 mM to 600 mM. By maintaining the salt concentration within this range, the stability of LRG can be further improved.

[0044] As the kind of salt contained in the LRG composition of the present invention, any known salt can be used, for example, metal salts (such as sodium salts, potassium salts and magnesium salts) and non-metallic salts (such as ammonium salts and amine salts) can be used. It is preferred to use a metal salt, and more preferably a sodium salt. In order to provide a sodium salt, sodium chloride is most preferably used. More than one salt can also be used in combination.

[0045] (Leucine-rich α2-glycoprotein composition)

[0046] In the present specification, the leucine-rich α2 glycoprotein composition (hereinafter sometimes referred to as the LRG composition) refers to a solution containing LRG for measuring LRG. The LRG composition of the present invention can be suitably used as a calibration sample solution for LRG measurement. In the present specification, the calibration sample solution refers to a sample solution containing a certain concentration of a substance to be measured, which is used to accurately measure the substance to be measured, and the standard substance, calibrator, control, internal standard substance, etc. are the calibration sample solution. The form for providing the LRG composition of the present invention is a form in a solution state prepared in advance by mixing LRG and a solvent having a pH of 7.0 to 9.3. Another form for providing the LRG composition of the present invention is a form in which LRG and a solvent are prepared separately and the two are mixed into a solution state before use.

[0047] The concentration of LRG in the LRG composition of the present invention may be 0.1 to 1000 μg / mL, 0.1 to 100 μg / mL, 1 to 100 μg / mL, 1 to 50 μg / mL or 1 to 30 μg / mL, although the concentration is not limited to this range.

[0048] In this regard, "using a leucine-rich α2 glycoprotein composition" in the method for measuring leucine-rich α2 glycoprotein means that the leucine-rich α2 glycoprotein composition is used for accurately measuring leucine-rich α2 glycoprotein, and means, for example, that the leucine-rich α2 glycoprotein composition is used as a calibration sample solution (standard substance, calibrator, control, internal standard substance, etc.).

[0049] There is no particular limitation on the composition of the LRG composition of the present invention, except that the LRG composition contains LRG and has a pH of 7.0 to 9.3, as long as the effect of the present invention is not impaired and the use of LRG is not hindered. When LRG is measured by an immunological assay method, the effect of the present invention should not be impaired, and all or part of the reactions constituting the assay system, such as antigen-antibody reaction, labeling reaction with biotin / avidin detection, and enzymatic reaction, should not be blocked. Components commonly used in immunological assays can be appropriately selected and used depending on the purpose, for example, buffers (e.g., acetic acid, citric acid, phosphoric acid, HEPES, MES, Tris, glycine, boric acid, carbonic acid and Good's buffer), components that suppress nonspecific reactions (nonionic surfactants, such as Tween 20 and Triton X-100, etc.), components that promote antigen-antibody reactions (polymers, such as polyethylene glycol, polyvinyl pyrrolidone and phospholipid polymers, etc.), glycoproteins or peptides other than LRG (albumin, casein, etc.), amino acids, sugars (sucrose, cyclodextrin, etc.), preservatives (sodium azide, ProClin 950, etc.), etc.

[0050] The LRG composition of the present invention can be stored in a known container. The material of the storage container that can be used for the LRG composition of the present invention may be polypropylene, polystyrene, or glass, although the material is not limited to these materials. The form of the storage container may be hard or soft, and examples include ampoules, vials, soft bags, injection-type containers, and the like.

[0051] LRG can be produced by cells expressing LRG. Cells expressing LRG can be obtained by transforming a host with an expression vector containing DNA encoding LRG. The host cell can be a microbial cell, an insect cell, a mammalian cell, etc., and can be a prokaryotic cell or a eukaryotic cell. As mammalian cells, for example, HepG2 cells, HEK293 cells, HeLa cells, human FL cells, monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter referred to as CHO cells), dhfr gene knockout CHO cells, mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat H4IIE-C3 cells, rat GH3 cells, etc. can be used.

[0052] The plasmid obtained in the above manner can be introduced into a host cell by conventional genetic engineering methods. The transformant can be cultured by a general method for culturing microorganisms or culturing insect cells or mammalian cells. The LRG protein can be obtained by a combination of methods generally used for isolating / purifying ordinary proteins.

[0053] In the specification, "storage stabilization" or "improved stability" means that since most of the LRG contained in the solution containing LRG is maintained for a long time without decomposition or structural change, the initial LRG value of the solution is not much different from the measured value after storage. More specifically, for example, this means that since 75% or more of the LRG contained in the solution containing LRG is maintained for 29 days without decomposition or structural change, the LRG value of the solution measured after storage at 37°C for 29 days is 75% or more of the initial value. After storage at 37°C for 29 days, the measured LRG value of the LRG composition of the present invention is preferably 80% or more based on the initial amount, more preferably 90% or more, further preferably 95% or more, and most preferably 96% or more. The LRG composition of the present invention has long-term stability also at a high temperature of 37°C, but LRG compositions stored at low temperatures or room temperature are not excluded from the scope of the present invention.

[0054] (Biological samples for LRG determination)

[0055] The biological sample used for measuring LRG is not particularly limited as long as LRG can be measured, and may be blood, serum, plasma, cerebrospinal fluid (CSF), urine, feces, etc. Serum or plasma is preferably used, and serum is further preferably used. The organism or subject is not particularly limited as long as the organism or subject is a mammal and includes humans or animals (e.g., monkeys, dogs, cats, mice, guinea pigs, rats, hamsters, horses, cattle, and pigs), preferably humans. If necessary, the biological sample may be subjected to appropriate pretreatment.

[0056] (Assay kit for leucine-rich α2-glycoprotein)

[0057] Using the kit for measuring leucine-rich α2 glycoprotein of the present invention (hereinafter sometimes referred to as LRG assay kit), LRG can be accurately measured using an LRG composition. An example of an LRG assay kit is a kit using an immunological method. The LRG assay kit of the present invention may include reagents for measuring the LRG concentration of a human body by an immunological method. Immunological methods include ELISA, enzyme immunoassay, surface plasmon resonance, latex agglutination immunoassay (LTIA), chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescent antibody technology, radioimmunoassay, immunoprecipitation, protein blotting, immunochromatography, high performance liquid chromatography (HPLC), and the like. The immunological method is preferably a latex agglutination immunoassay (LTIA).

[0058] The LRG assay kit of the present invention can be used to diagnose autoimmune diseases, tuberculosis, tumors, inflammatory diseases and inflammatory bowel diseases (ulcerative colitis and Crohn's disease).

[0059] The LRG assay kit of the present invention may further include a user guide, etc. The kit may further include any constituent elements such as a buffer, a stabilizer, a sample diluent, a pH adjuster, and a reaction container.

[0060] (Storage stabilization method of LRG composition)

[0061] The storage stabilization method for the LRG composition of the present invention includes the step of contacting the leucine-rich α2 glycoprotein with a solvent having a pH of 7.0 to pH 9.3. The solvent is not particularly limited as long as the pH is 7.0 to 9.3. The solvent may contain a buffer such as HEPES, MES, CHES, and Tris, and the concentration of the buffer may be 1 to 1000 mM. In addition to adding LRG to a solvent having a pH of 7.0 to 9.3, "contacting" also includes adjusting the pH of a solution containing LRG that is not 7.0 to 9.3 to 7.0 to 9.3.

[0062] Next, the present invention is specifically described with reference to Examples, but these Examples do not limit the scope of the present invention. In this specification, % means weight % unless otherwise specified.

[0063] Example

[0064] [Example 1] Examination of the change of LRG amount over time in solutions with different pH values ​​1

[0065] After preparing LRG standard antigen solutions with different pH values, a preservation test was performed, and the change in the LRG amount of the LRG standard antigen solution over time was examined. Therefore, the effect of pH on the preservation stability of LRG was examined. The LRG used was produced using CHO (Chinese hamster cells). Specifically, LRG was prepared by the following steps: introducing the human LRG gene into CHO-K1 cells using a plasmid, culturing the CHO-K1 cells into which the LRG gene was introduced in a serum-free medium, and recovering the human LRG recombinant protein from the culture supernatant.

[0066] The LRG standard antigen solution having the composition of Table 1 below was prepared, and the pH value was controlled to 7.5 or 8.0 using 4N sodium hydroxide solution. The LRG standard antigen solution was stored at 37°C for 29 days, and the change of LRG amount over time was observed. The sample composition and experimental results are shown in Tables 1 and 2 and Figure 1 middle.

[0067] [Table 1]

[0068] Element concentration Bovine serum albumin (BSA PF) 1％ HEPES 50mM Sodium chloride 150mM TWEEN 20 0.01％ EDTA3Na 5mM ProClin950 0.05％ Purified water Necessary amount LRG 10.0 μg / mL

[0069] [Table 2]

[0070] Sample No. pH of LRG standard antigen solution Measured value after 29 days (initial measured value is 100%) 1 7.5 81％ 2 8.0 93％

[0071] After 29 days of storage at 37°C, the measured value of the LRG standard antigen solution adjusted to pH 8.0 was 93% of the initial LRG amount. The measured value of the LRG standard antigen solution adjusted to pH 7.5 was 81% of the initial LRG amount. Therefore, it was shown that the storage stability of LRG was excellent when the pH of the LRG standard antigen solution was 8.0 instead of 7.5.

[0072] [Example 2] Examination of the change in LRG amount over time in solutions with different salt concentrations

[0073] In sample No. 2 prepared in Example 1, the concentration of sodium chloride was changed from 150 mM to 500 mM, and a storage test was performed. Thus, the effect of salt concentration on the storage stability of LRG was examined. The sample composition and experimental results are shown in Tables 3 and Figure 2 middle.

[0074] [Table 3]

[0075] Sample No. Salt concentration (mM) Measured value after 29 days (initial measured value is 100%) 2 150 93％ 3 500 98％

[0076] The results showed that the LRG standard antigen solution with a salt concentration of 500 mM can improve the storage stability of LRG compared with the LRG standard antigen solution with a salt concentration of 150 mM. After 29 days of storage at 37°C, the measured value of the LRG standard antigen solution with a salt concentration of 150 mM was 93% of the initial LRG amount, while the measured value of the LRG standard antigen solution with a salt concentration of 500 mM was 98% of the initial LRG amount after 29 days of storage at 37°C.

[0077] [Example 3] Examination of the change of LRG amount over time in solutions with different pH values ​​2

[0078] A LRG standard antigen solution having the same composition as the sample prepared in Example 1 was prepared, except that the buffer shown in Table 4 below was used and the pH was adjusted to 6.0, 7.0, 7.5, 8.0, 8.5, 9.0 or 9.5. The LRG standard antigen solution was stored at 37°C for 29 days, and the change in LRG amount over time was observed. The sample composition and experimental results are shown in Tables 4 and Figure 3 middle.

[0079] [Table 4]

[0080] Sample No. Buffer pH Measured value after 29 days (initial measured value is 100%) 4 MOPS 6.0 52％ 5 HEPES 7.0 75％ 6 HEPES 7.5 83％ 7 HEPES 8.0 100％ 8 HEPES 8.5 100％ 9 CHES 9.0 96％ 10 CHES 9.5 67％

[0081] From the above results, it was shown that when the pH of the LRG standard antigen solution was adjusted to 7.0 to 9.3, the storage stability of LRG was improved.

[0082] Industrial Applicability

[0083] The leucine-rich α2-glycoprotein composition of the present invention has excellent storage stability. Therefore, when the leucine-rich α2-glycoprotein composition of the present invention is used as a calibration sample, LRG in a biological sample can be accurately quantitatively measured, and a disease can be accurately diagnosed.

Claims

1. A calibration sample solution for measuring leucine-rich α2 glycoprotein, comprising leucine-rich α2 glycoprotein, wherein the pH of the calibration sample solution is 7.8 to 9.0, the salt concentration of the calibration sample solution is 300 mM to 600 mM, and the salt is a sodium salt, a potassium salt, or a magnesium salt.

2. The calibration sample solution according to claim 1, wherein the sodium salt is sodium chloride.

3. A method for determining leucine-rich α2 glycoprotein for non-diagnostic purposes, comprising: The calibration sample solution according to claim 1 or 2 is used.

4. A kit for measuring leucine-rich α2-glycoprotein, comprising the calibration sample solution according to claim 1 or 2.

5. A method for stabilizing the storage of leucine-rich α2 glycoprotein, comprising: contacting the leucine-rich α2 glycoprotein with a solvent having a pH of 7.8 to 9.0, The salt concentration of the solvent is 300 mM to 600 mM, and the salt is a sodium salt, a potassium salt or a magnesium salt.

6. The method for storage stabilization of leucine-rich α2 glycoprotein according to claim 5, wherein the sodium salt is sodium chloride.

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