JP3196123B2 - Cellulose acetate support for electrophoresis - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a support for electrophoresis comprising a microporous polymer membrane, and more particularly to an electrophoresis support containing cellulose acetate as a main component used for fractionating serum proteins. The present invention relates to a support for electrophoresis.

[0002]

BACKGROUND OF THE INVENTION Fractionation of serum proteins is carried out in the field of clinical examination. For this purpose, electrophoresis is carried out using a polymer microporous membrane containing a polymer such as cellulose acetate as a main component. Supports are now widely used in clinical tests in hospitals and the like. As a means for fractionating serum proteins by electrophoresis using a cellulose acetate membrane, the cellulose acetate membrane is immersed in a buffer solution, and then the serum is applied to the membrane surface and a DC voltage is applied to the protein to remove the protein. After electrophoresis and fractionation, the protein is stained. In this case, a veronal buffer having a pH of 8.6 is usually used as the buffer.
Small and separation especially separation of albumin fraction and alpha ₁ over globulin fraction of each fraction is deteriorated). In the electrophoresis phenomenon, protein components in an electric field migrate toward the anode, while protein components with low isoelectric point (albumin fraction) are attracted to the anode and protein components with high isoelectric point (γ-globulin fraction). )
Is attracted to the cathode. The electrophoretic images on the support thus divided were albumin, α ₁
Over globulin, α ₂ over globulin, β over globulin, γ
Globulin and usually divided into 5 fractions. Then, as a primary screening, the percentage of each fraction is accurately measured first to diagnose the presence, type and extent of the disease, and a more rigorous protein test is carried out for a problematic result. Cellulose acetate support for electrophoresis previously proposed by the present applicant (Japanese Patent Application Laid-Open No. 1-227055) (hereinafter referred to as "support of prior application")
) Is intended to contribute to such a fractionation method, so that the application point is completely separated from the fractionation zone and the sample residue is generated so that the accurate fractionation percentage of serum protein can be measured. The purpose of the present invention is to provide a support for electrophoresis mainly comprising cellulose acetate. However conventional cellulose - Suasete - DOO support, of course, still nephropathy in this prior support - Ze syndrome, separation of alpha ₂ over globulin fraction and β over globulin fraction in the serum of such hyperlipemia is not clear, Often this was a source of measurement error. Particularly in such serum, low density lipoprotein (β
Lipoproteins), which will result in different fractionated positions. For example, α ₂ over globulin fraction and the β
Over globulin fraction to occur fraction of β Ripo protein, further the fraction of β Ripo protein located between alpha ₂ over globulin fraction and β over globulin fraction varies depending on the content of β Ripo protein. That is, the content of β Ripo protein contained as the alpha ₂ over globulin fraction less, and high content alpha
_An independent fraction is generated between the _2- globulin and β-globulin fractions. The α ₂ over globulin fraction behind in the hand -
In some cases the ring occurs alpha ₂ over globulin fraction and β over globulin fraction becomes one of fractionation. For this reason,
At present, there is a strong demand for the appearance of a support for electrophoresis free of such defects.

[0003]

SUMMARY OF THE INVENTION In accordance with the above-mentioned demands, the present invention has been studied as a result of an intensive study to find that fatty acid esters and oxyethylene derivatives of polyhydric alcohols emulsify and solubilize oils and esters. In view of the fact, it was conceived to clarify the 5-fraction of serum of nephrotic syndrome and hyperlipidemia by these actions. Then the low-density lipoprotein (beta Ripo protein) of the containing alpha ₂ over globulin fraction due to the amount of the beta over globulin fraction defective separation of the serum, cellulose - Suasete - bets on microporous film composed mainly of It is an object of the present invention to provide a cellulose acetate support for electrophoresis which makes each fraction clear by adding and containing a fatty acid ester at a constant weight ratio.

[0004]

According to the present invention, there is provided a microporous membrane containing cellulose acetate as a main component, having an electroosmosis coefficient of less than 0 to -3 m.
m, which contains at least one polyoxyethylene sorbitol fatty acid ester as an additive in a weight ratio of 0.3 to 1.9% with respect to the polymer. This is a cellulose acetate support for electrophoresis, and the additive can be added internally to the dove or externally after film formation. Polyoxyethylene sorbitol tetraoleate is the most effective and desirable polyoxyethylene sorbitol fatty acid ester as the above additive.

[0005]

[Description of Manufacturing Process] The manufacturing process will be described below. To form a microporous membrane, a phase separation method, one of the methods for manufacturing a polymer microporous membrane, is used. That is, cellulose acetate (diacetate, triacetate) is used as a lipophilic solvent, a poor solvent,
A dope prepared by dissolving in a non-solvent mixed solvent is cast and dried. The above-mentioned lyophilic solvents include acetone, ethyl acetate, methylene chloride and the like, the poor solvents include tetrahydrofuran, methanol and ethanol, and the non-solvent is water in many cases. Cellulose acetate is dissolved in these mixed solvents to a concentration of 1 to 20% as described above. Further, "Reodol 430, 440, 460" (trademark of Kao Products) is used as a polyoxyethylene sorbitol tetraoleate as an additive.
Is used. The general formula of polyoxyethylene sorbitol tetraoleate is as follows: Polyoxyethylene sorbite represented by the above general formula
Letetraoleate includes (CH ₂ CH ₂ O) n
= 30, most preferably Leodor 430 (trademark of Kao products). The method of addition is to add internally to the dove or
Alternatively, it may be externally added after film formation, and the weight ratio to the polymer is optimally around 0.7% in the range of 0.3% to 1.9%. As described above, an electrophoresis support having no separation failure due to the content of the low-density lipoprotein (β-lipoprotein), that is, accurate measurement of fractionation percentage is completed.

[0006]

[Comparative Experimental Examples] The present invention will be described in more detail with reference to the following experimental examples. * The weight ratio of leotole 430 to polymer is as follows. Comparative Example 1 Leotole 430 to polymer 0.2% Example 1 Leotole 430 to polymer 0.3% Example 2 Leotole 430 0.7% relative to polymer Example 3 Leoitol 430 versus 1.9% polymer Comparative Example 2 2.0% Leoitol 430 versus 2.0% Comparative Example 3 Conventional product

A uniform dope was prepared with the above composition, and this solution was cast on a glass plate to a thickness of 1 mm, left in an atmosphere at a temperature of 25 ° C. and a humidity of 65% for 20 minutes, and formed from the glass plate. The film was peeled off and dried in an oven drier at 60 ° C. for another 1 hour. Each of the microporous membranes thus obtained had a dry thickness of about 150 μm and an average pore diameter of 0.45.
Physical properties in the range of ２．０2.0 μm. This film is 12 × 6
cm, and immersed in veronal / veronal sodium buffer (pH 8.6 0.06 mol), and then applied 10 serum samples each of 5 types of serum of nephrotic syndrome and hyperlipidemia. (Application position 2: 8 from the cathode) Electrophoresis was performed at a current of 0.5 mA / cm for 30 minutes. Next, the membrane was immersed in a 0.8% Ponso-3R (trademark of Wako Pure Chemical Industries) solution dissolved in a 6% aqueous solution of trichloroacetic acid for 2 minutes, stained, and further washed with a 2% aqueous solution of acetic acid five times for 1 to 2 minutes. Decolorized.
As a result, comparative results as shown in Table 1, Table 2, Table 3, Table 4, and Table 5 were obtained. The n number in the table is the result of measuring 10 electrophoreses, electrophoresis images after staining, decolorization and drying with a densitometer (densitometer: ADC-25EX manufactured by Kayagaki Seisakusho).
It is the number of samples that have been fractionated, and each value is a value based on the number of samples.

[0008]

[Table 1]

[0009]

[Table 2]

[0010]

[Table 3]

[0011]

[Table 4]

[0012]

[Table 5]

The following facts have been found from the above tables. That Comparative Example 1 - a separation between (Reoto Bu Le 430 vs. 0.2% polymer), the alpha ₂ over globulin fraction and β over globulin fraction defective, Comparative Example 2 (Reoto Bu - Le 430 versus polymer 2.0 %) Is 5
Each band of the fraction was indistinct. On the other hand, Example 1 (leotole 430 to polymer 0.3%), Example 2 (leotole 430 to polymer 0.7%) and Example 3 (leotole 430 to polymer 1.%). (9%), the resolution was good and the band was clear.
7%) was the best and clearest. From this fact, it was found that adding 0.3 to 1.9% of the surfactant Reodor 430 has a great effect. Note was subjected to electrophoresis on even the樣the conventional film (Comparative Example 3) For comparison, the separation between the alpha ₂ over globulin fraction and β over globulin fraction poor, were bands unclear. The electrophoretic image of Example 1 was scanned with a densitometer, and the result of continuous measurement of the absorbance of each fractionation section was graphically displayed as shown in FIG. 1. The electrophoretic image corresponding to FIG. 1 is a photograph of FIG. As shown in FIG. Further, the electrophoretic image of the conventional membrane of Comparative Example 3 was scanned with a densitometer, and the results of continuous measurement of the absorbance of each fractionation section were graphically displayed as shown in FIG. The electrophoresis image is as shown in the photograph of FIG.

[0014]

As described above, the present invention provides a fatty acid ester of polyhydric alcohol and oxyethylene having an emulsifying and solubilizing action of esters and the like on a microporous membrane containing cellulose acetate as a main component. By containing a derivative as an additive, the serum of nephrotic syndrome and hyperlipidemia
It succeeded in further improving the separation ability of fractions and band clarity, and accurately inspected for the presence, type, and degree of disease.
Provided is a cellulose acetate support for electrophoresis which is extremely effective for diagnosis.

[Brief description of the drawings]

FIG. 1 is a graph obtained by scanning an electrophoretic image of Example 1 with a densitometer and continuously measuring the absorbance of each fractionation part.

FIG. 2 is a graph in which the electrophoresis image of Comparative Example 3 is scanned with a densitometer and the absorbance of each fraction is continuously measured.

FIG. 3 is a photograph showing an electrophoretic image of a support of Example 1.

FIG. 4 is a photograph showing an electrophoretic image of a support of Comparative Example 3.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 27/447 CA (STN) JICST file (JOIS) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A microporous membrane containing cellulose acetate as a main component and having an electroosmotic coefficient of less than 0 to minus 3
mm, and at least one polyoxyethylene sorbitol fatty acid ester as an additive in a weight ratio of 0.3 to 1.9% relative to the polymer. Cellulose acetate for electrophoresis
G support. 2. The cellulose acetate support for electrophoresis according to claim 1, wherein the polyoxyethylene sorbitol fatty acid ester as an additive is polyoxyethylene sorbitol tetraoleate.