JPH09169799A - Lipoprotein composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a lipoprotein composition with its characteristics retained stably for a long period, widely applicable to the areas of medical care and biology, etc., and among others, directly usable as a reference substance for diagnoses or as an antigen for immunization in preparing antibodies such as polyclonal and monoclonal ones. SOLUTION: This lipoprotein-contg. composition is obtained by mixing a buffer solution at pH6.0-9.5 containing neither phosphoric acid not citric acid with 1-20wt.% of a disaccharide and/or monosaccharide and 0.05-10mM of a chelating agent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a long-term stable lipoprotein composition.

[0002]

2. Description of the Related Art Low-density lipoprotein (hereinafter referred to as "LDL") is a serum lipoprotein fraction having a specific gravity in the range of 1.063 to 1.125 and transports fat-soluble substances to various tissues in the body through blood. Have a job. However, it is known that excessive presence in blood causes arteriosclerotic diseases such as myocardial infarction. In addition, the lipoprotein (a) (hereinafter referred to as “Lp (a)”), which is a genetic variant of LDL, is LD
It has an L-like structure with a specific protein Apo (a) bound to the nucleus, and this lipoprotein (hereinafter sometimes simply referred to as "lipoprotein") is also an independent arteriosclerotic disease. Recently, it has been attracting attention as a risk factor for. The above LDL, Lp (a) and the like are collectively referred to as LDL-like lipoproteins. Measuring the blood levels of these LDL-like lipoproteins is useful for preventing arteriosclerotic diseases,
It is extremely useful for diagnosis, and the immunological assay method using a polyclonal antibody occupies the mainstream at present. In recent years, it has been desired to develop an assay system using a monoclonal antibody having higher specificity. For that purpose, it is necessary to secure a highly purified purified preparation in a highly stable state. In general, the above substances have been obtained by fractionating serum by ultracentrifugation and then isolating it by column chromatography.

[0003]

However, when the above LDL-like lipoprotein is left in a solution for a long period of time, it causes aggregation (intermolecular aggregation) and is likely to be denatured. I'm holding. Currently sugars (glucose, sucrose, etc.)
Although measures have been taken to suppress aggregation by adding a small amount of, the definitive stabilization method has not yet been established. Therefore, it is difficult to store the LDL-like lipoprotein while maintaining a stable state for a long period of time,
It has been a major obstacle to the production of monoclonal antibodies.

As described above, LDL or L similar thereto
At present, compositions containing DL-like lipoproteins have a short life span and are limited in marketability. Therefore, the appearance of a lipoprotein composition that is excellent in long-term stability and that can be maintained for a long time while maintaining the specificity of the contained protein has been awaited. INDUSTRIAL APPLICABILITY The present invention has a property that the lipoprotein contained in the lipoprotein composition is stably maintained for a long period of time, and can be used in a wide range of fields such as medical care and biology. In particular, an antibody such as a polyclonal antibody or a monoclonal antibody is used. The present invention provides a lipoprotein composition which can be used as it is as an immunizing antigen at the time of preparation and as a standard substance in diagnosis.

[0005]

The inventors of the present invention have conducted extensive studies to solve the above-mentioned problems, and as a result, as a base solution for lipoprotein composition solutions such as LDL and Lp (a), a physiological solution was prepared. Saline, phosphate buffer saline (hereinafter referred to as “PBS”), and citrate buffer cause aggregation when used as a base,
It was found to denature easily. As a result of further studies, it was found that stability can be maintained for a long period of time while retaining the specificity of lipoproteins, if a specific amount of saccharides such as sucrose is added as a stabilizer based on a specific buffer solution. I found it. Further, it was confirmed that the use of the composition of the present invention significantly improved the production efficiency even when producing a monoclonal antibody, and the present invention was completed.

That is, the present invention relates to a lipoprotein-containing composition containing 1 to 1 of at least one of a disaccharide and a monosaccharide in a buffer solution containing neither phosphoric acid nor citric acid and having a pH of 6.0 to 9.5. 20% by mass, and a chelating agent of 0.05 to 10 mM are mixed, which is a lipoprotein composition.

[0007] The main component, lipoprotein, is obtained by ultracentrifuging the serum component, and the corresponding density fraction is isolated by gel filtration or by column chromatography such as affinity chromatography. The obtained lipoprotein such as LDL or Lp (a) is used as a monitor for disease diagnosis, a standard substance, and an immunogen at the time of preparing an antibody.

[0008]

DETAILED DESCRIPTION OF THE INVENTION The present invention will be described in detail below. The lipoprotein of the present invention is chylomicron,
Very low density lipoprotein (hereinafter referred to as "VLDL"),
Examples thereof include LDL and Lp (a), and high density lipoprotein (hereinafter referred to as “HDL”) and the like can be included therein. Preferably, chylomicron, VLDL, L
DL, Lp (a), and particularly preferably LDL, L
p (a). A method for separating and purifying lipoprotein can be obtained by a known method such as disclosed in JP-A-4-293496, and an example thereof will be described.

LDL or Lp (a) is obtained from human-derived serum or plasma. It is desirable that the raw materials used be as fresh as possible and stored sufficiently carefully. Separation and purification of lipoproteins is performed using the ultracentrifugation method, and generally, "sequential ultracentrifugation method" in which salts and solutions are sequentially added to give a corresponding density to adjust the specific gravity, or two or more solutions with different specific gravities are used. A "density gradient method" in which a substance is added and subjected to a density gradient for separation is known. The lipoprotein in the present invention is purified by sequentially adding solid KBr to fresh human serum to adjust the specific gravity, and subjecting the specific gravity fraction to column treatment. LDL is obtained by treating a fraction having a specific gravity of 1.019 to 1.063 with an anti-Lp (a) affinity column,
Lp (a) is HDL with a specific gravity of 1.063 to 1.125.
A purified sample is obtained by treating the two fractions with a gel filtration column.

The buffer solution used in the present invention is a buffer solution containing neither phosphoric acid nor citric acid and having a pH of 6.0 to 9.5. The buffer solution that can be used in the present invention includes tris-hydrochloric acid, tris-maleic acid and other tris buffer solutions, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (hereinafter referred to as HEPE).
SEP) -HEPES buffer such as NaOH, bis (tris) propane (hereinafter also referred to as BTP) buffer,
N-2-hydroxyethylpiperidine-N'-propanesulfonic acid (hereinafter also referred to as EPPS) buffer, 2- (N-morpholino) ethanesulfonic acid (hereinafter also referred to as MES) buffer, 3- (N-morpholino) ) Propane sulfonic acid (hereinafter M
OPS) buffer, piperazine-N, N′-bis (2-ethanesulfonic acid) (hereinafter also referred to as PIPES) buffer, tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (hereinafter also referred to as TAPS) Buffer), N-
Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (hereinafter, also referred to as TES) buffer solution, N-tris (hydroxymethyl) methylglycine (hereinafter, TRIC)
INE) buffer, veronal buffer, borate buffer, glycine-HCl, imidazole-HCl, collidine-HCl, N-heptylmorpholine-HCl, triethanolamine-HCl, triethanolamine-Na.
OH, pyrophosphate-HCl, methylimidazole-HC
1, etc., but preferably Tris-salts, Tris buffer such as Tris-maleic acid, HEPES-NaOH.
HEPES buffer such as Tris-
Hydrochloric acid, tris-maleic acid, HEPES-NaOH.

The concentration of the above buffer solution is 2 to 100 mM, preferably 5 to 50 mM, more preferably 5 to 20 mM.
Particularly preferably, it is 5-18 mM. Also pH at 4 ° C
Is 6.0 to 9.5, preferably pH 6.5 to 9.0, more preferably pH 6.5 to 8.5, and particularly preferably pH 6.5 to 8.0.

In the composition of the present invention, saccharides have a function of suppressing aggregation of lipoproteins and contribute to stabilization of lipoproteins which denature over time. The saccharides are glucose, monosaccharides such as fructose and saccharose, disaccharides such as maltose are used, preferably monosaccharides having a ketone group such as disaccharides or fructose, more preferably disaccharides, specifically, Saccharose, which has high solubility and is economically inexpensive, is preferably used. As these saccharides, either one of monosaccharides and disaccharides may be used alone, or both may be used as a mixture.
In addition, plural kinds of saccharides selected from monosaccharides and disaccharides may be mixed and used. Of these, a monosaccharide having an aldehyde group such as glucose may interact with an amino group of the protein to cause glycation modification when left with a lipoprotein for a long period of time. If the amount of the above-mentioned saccharides is too large, the viscosity of the composition liquid becomes high, which causes troubles in handling. Therefore, it is about 20% by mass or less, preferably 10% by mass, based on the total composition of the present invention.
The following is good. Further, if the amount is too small, the above effect is not exhibited, so the lower limit is 1% by mass, preferably 5% by mass based on the above criteria.

The chelating agent used together with the above-mentioned saccharide in the composition of the present invention has a function of masking metal ions in the solution and suppressing oxidative denaturation of lipoprotein. Ethylenediamine (E
DA), ethylenediaminetetraacetic acid (EDTA), ethyleneglycolbis (2-aminoethylether) tetraacetic acid (EGTA), dimethylglyoxime, acetylacetone, glycine, etc., but most preferred are
And EDTA and EGTA that do not denature lipoproteins
Is good. Since the chelating agent is effective even in a small amount, 0.05 to 10 mM, preferably 0.5 to 5 mM
Is added. If it is less than 0.05 mM, the effects of the present invention cannot be sufficiently achieved. Further, even if it exceeds 10 mM, the effect of the present invention cannot be further enhanced.

The addition of these chelating agents is 0.05 mM to 10 mM with respect to the buffer solution or the lipoprotein composition.
The total amount of the chelating agent and the unreacted chelating agent added in the composition was 0.05 mM to 10 mM.
Mean contained in mM. In the addition of the saccharide and the chelating agent of the present invention, the saccharide and the chelating agent can be added in any order to the buffer solution or the buffer solution containing lipoprotein. Specifically, either or both of a saccharide and a chelating agent are added to the buffer solution of the present invention in advance, and the buffer solution is used for purification of lipoprotein, or by dialysis etc. After containing the lipoprotein, saccharides and chelating agents may be adjusted to have the composition of the present invention. Further, after adding lipoprotein to the buffer solution of the present invention, a saccharide and a chelating agent may be added so as to have the composition of the present invention.

Preferably, the lipoprotein-containing solution is dialyzed with a buffer solution to which a chelating agent is added in an amount of 0.05 mM to 10 mM in advance so as to contain the lipoprotein, and the saccharide is contained therein in an amount of 1 to 20% by mass. Is added to obtain the lipoprotein composition of the present invention. The lipoprotein composition of the present invention may contain various components other than the above-mentioned components. Examples of components that can be added include salts such as NaCl, KCl and KBr, proteins such as serum albumin, preservatives such as sodium azide (NaN ₃ ) and parabens. These components that can be added can be included in the composition of the present invention at any time. Although the lipoprotein composition stabilized as described above is used in the fields of medicine and biology, it is particularly preferably used as an antigen for immunization at the time of preparing an antibody such as a polyclonal antibody or a monoclonal antibody and a standard substance in diagnosis. .

[0016]

EXAMPLES The effects of the present invention will be described more specifically with reference to the following examples. Examples 1 to 8 and Comparative Example 1 below
A lipoprotein composition was prepared as in. Example 1 Solid KBr was added to fresh human serum to give a specific gravity of 1.06.
It was adjusted to 3, and ultracentrifuged at 150,000 G for 20 hours or more to obtain an LDL fraction. The separated LDL fraction is anti-Lp
(A) A purified LDL preparation was obtained by applying it to an affinity column using a polyclonal antibody as a ligand to remove contaminating Lp (a). Purified LDL is 1 mM-ED
10 mM Tris-HCl buffer (pH 7.
2) It was dialyzed with 1000 times amount for 20 hours or more, and saccharose was added so as to be 10% by mass after completion to obtain a lipoprotein composition. Example 2 10 mM Tris-HCl buffer (pH 7.
A lipoprotein composition was obtained by the same method except that 2) was changed to 15 mM Tris-HCl buffer (pH 7.2).

Example 3 A lipoprotein composition was obtained by the same method except that sucrose in Example 1 was changed to maltose. Example 4 A lipoprotein composition was obtained in the same manner as in Example 1, except that 10% by mass sucrose was changed to 15% by mass sucrose and 1 mM-EDTA was changed to 1 mM-EGTA. Example 5 10 mM Tris-HCl buffer (pH 7.
2) 10 mM HEPPES-NaOH buffer (pH
A lipoprotein composition was obtained in the same manner except that the composition was changed to 7.2).

Example 6 A lipoprotein composition was obtained by the same method except that sucrose in Example 1 was changed to fructose. Example 7 A lipoprotein composition was obtained in the same manner as in Example 1, except that sucrose was changed to glucose. Example 8 A lipoprotein composition was obtained in the same manner as in Example 1 except that sucrose was replaced with galactose.

Comparative Example 1 In Example 1, 10 mM Tris-HCl buffer solution (pH 7.2) containing 1 mM-EDTA was added to 10 mM PBS.
A lipoprotein composition was obtained in the same manner except that the buffer solution (pH 7.2) was changed and 10% by mass sucrose was not added. Comparative Example 2 In Example 1, 10 mM Tris-HCl buffer (p
A lipoprotein composition was obtained in the same manner except that H7.2) was changed to 10 mM PBS buffer (pH 7.2). Comparative Example 3 In Example 1, 10 mM Tris-HCl buffer (p
A lipoprotein composition was obtained by the same method except that H7.2) was changed to 10 mM citrate buffer (pH 7.2).

Comparative Example 4 A lipoprotein composition was obtained in the same manner as in Example 1 except that 10% by mass of sucrose was not added. Comparative Example 5 A lipoprotein composition was obtained in the same manner as in Example 1 except that 1 mM-EDTA was not added.

The stability of the lipoprotein compositions prepared as in Examples 1-8 and Comparative Examples 1-5 above was evaluated. The stability was evaluated by the following polyacrylamide disc gel electrophoresis (PAGE) method and agarose gel immunoelectrophoresis method. (Polyacrylamide disc gel electrophoresis (PAG
Method for Evaluating Stability According to E)) The lipoprotein compositions obtained in Examples and Comparative Examples were analyzed with a commercially available polyacrylamide disc gel analysis kit (Tsuko, "Lipophor") to evaluate their storage stability. . In this method, a sample is pre-stained with a Sudan Black solution that specifically stains lipids, treated at room temperature for 1 hour, and then applied to a disc. Further, a constant current of 10 mA is applied to each disk, and 2
After running for 0 minutes, the sample was identified or the purity was evaluated based on the separation pattern.

(Stability (antigenicity) evaluation method by agarose gel immunoelectrophoresis) Whether the lipoprotein compositions obtained in Examples and Comparative Examples were subjected to immunoelectrophoresis to retain their antigenicity I'm sure. 1% agarose gel (“Universal gel 8” manufactured by Corning Incorporated, USA)
Then, 5 μl of 1 mg / ml purified LDL was added thereto, and electrophoresis was performed in a barbital buffer (manufactured by Daiichi Pure Chemicals Co., Ltd.) at 90 V for 50 minutes. After the completion of the electrophoresis, 20 μl of 1 mg / ml anti-apoB polyclonal antibody was added to both sides of the electrophoresis gel and left at room temperature for 24 hours. The next day, excess protein was removed with physiological saline and Coomassie Brilliant Blue stain ("Page Blue 83" manufactured by Daiichi Pure Chemicals)
The antigen-antibody precipitation line was stained with. To evaluate the stability, the lipoprotein compositions of Examples and Comparative Examples were stored at 4 ° C. for 2 weeks to 9 months, and the stability was evaluated by the above method at each time.

The stability evaluation criteria are as follows. × -Antigen-antibody precipitation line disappeared within 2 to 4 weeks, PAGE
Electrophoresis pattern changes △ -The antigen-antibody precipitation line decreases to about 80% in 1-2 months, P
Change in AGE migration pattern ○ Antigen-antibody precipitation line decreased to about 80% in 2 to 6 months, P
AGE migration pattern changes ◇ -6 months, the antigen-antibody precipitation line is separated, PAGE migration pattern is separated into 2 ◎-9 months, the antigen-antibody precipitation line is 100%, PAGE
No change in the migration pattern

Table 1 shows the evaluation results by the above-mentioned polyacrylamide disc gel electrophoresis (PAGE) method and agarose gel immunoelectrophoresis method.

[0025]

[Table 1]

(Results of stability evaluation by polyacrylamide disc gel electrophoresis (PAGE)) Analysis results,
The purified LDL preparation was detected as a single band, and its migration pattern did not change even after 9 months. On the other hand, when a PBS buffer solution or a citrate buffer solution was used, aggregation occurred in a few weeks and the electrophoretic band disappeared. This proves that the purified LDL composition liquid of the present invention exhibits high stability.

(Results of Stability (Antigenicity) Evaluation by Agarose Gel Immunoelectrophoresis) As a result of analysis, the purified LDL stabilized by the present invention gave an immunoprecipitation line to the anti-apo B polyclonal antibody for 9 months. The same results were obtained by analysis using a standard that had passed. This indicates that the LDL composition solution of the present invention maintains the antigenicity of apoB, which is an LDL-specific protein, even after 9 months have passed. On the other hand, when PBS is used as the base, aggregation occurs in a few weeks, so that the sedimentation line disappears and the antigenicity is lost. This suggests that the composition liquid of the present invention exhibits excellent effects even when used as an antigen for antibody production.

[0028]

INDUSTRIAL APPLICABILITY By using the lipoprotein composition of the present invention, the characteristics of the lipoprotein contained therein can be stably maintained for a long period of time. INDUSTRIAL APPLICABILITY The lipoprotein composition of the present invention is used in a wide range of fields such as medical care and biology, and in particular, it can be used as it is as an immunizing antigen at the time of preparing an antibody such as a polyclonal antibody or a monoclonal antibody or a standard substance in diagnosis.

Claims (1)

[Claims] 1. A composition containing lipoproteins, wherein the composition contains neither phosphoric acid nor citric acid and has a pH of 6.
In a buffer solution of 0 to 9.5, 1 to 20 mass% of at least one of a disaccharide and a monosaccharide, and a chelating agent of 0.
A lipoprotein composition characterized by being mixed in an amount of 05 to 10 mM.