JP2567648B2 - Cellulose acetate support for electrophoresis - Google Patents

Description

TECHNICAL FIELD The present invention relates to an electrophoretic support, and particularly to an electrophoretic support containing cellulose acetate as a main component used for fractional electrophoresis of serum proteins. It is about the body.

(Prior Art) Serum protein fractionation by cellulose acetate membrane electrophoresis is now widely practiced as a routine test method for clinical tests in hospitals and the like. In this case, the serum protein of the sample is usually separated into five fractions of albumin, α ₁ -globulin, α ₂ -globulin, β-globulin, and γ-globulin, and the percentage fraction of each fraction is measured to determine the presence or absence of disease.
It is used as a material for diagnosing type, degree, etc. Therefore, it is extremely important to accurately measure the fractional percentage of the five fractions. However, in the case of the conventional cellulose acetate support, the sample application point (origin) overlaps with this fraction band or fraction part, and the sample residue remains at the application point even after migration,
It often caused an error in measurement.

For this reason, there has been a strong demand for the development of a support having no such disadvantages.

(Problems to be Solved by the Invention) The present invention meets these needs and eliminates the above-mentioned drawbacks.
In order to be able to measure the accurate fractional fraction of serum proteins,
The present invention intends to provide a new electrophoretic support mainly composed of cellulose acetate, in which the application point is completely separated from the fractionated zone and a sample residue is not generated.

In electrophoresis of serum proteins, it is necessary that the fractionation part is developed in the central part of the support as much as possible. However, for this purpose, the influence of the electroosmotic phenomenon of the support must be taken into consideration when determining the coating point.

The electro-osmotic coefficient of a general electrophoretic support currently sold in the market is measured by the method described in JP-B-55-31418, and it is found to be minus 20 mm to minus 4.5 m.
was in the m range. For example, some commercial products have an electro-osmotic coefficient of about -20 mm, and the optimum application point is 7:
Position 3 However, this application point overlaps with the α ₁ -globulin fraction. In other commercially available products, the electroosmotic coefficient is about −4.5 mm, and the optimum application point is at the position of 3: 7 from the negative electrode side. In this case, the application point is located at the end of γ-globulin.

As described above, almost all the conventional commercial products have the application point in the fraction band, and the presence of the sample residue at the application point is clearly recognized even after the electrophoresis. Therefore, the percentage of the fractionated protein cannot be accurately measured. It is impossible.

Recently, a cellulose acetate support with an electroosmotic coefficient of 0 has been marketed as "for isoelectric point fractionation". When serum protein electrophoresis was performed using this, the application point and the protein fractionation zone were separated. However, not only the amount of coating residue at the coating point is large and the percentage becomes inaccurate, but also this residue is easily misidentified as a fractionation part, so that it cannot be used for serum fractionation.

(Means for Solving the Problems) In order to improve these points, the inventors of the present invention have conducted extensive studies, and as a result, have found that the microporous membrane containing cellulose acetate as a main component has an electroosmotic coefficient of less than 0 to minus 3 mm.
, The average pore diameter is 0.2 to 5 μm, and the surface pore diameter of the sample application surface is 10 to 30 μm, so that the origin is completely separated from the protein fraction band, and after application, the application residue It was found that a support in which each of the fractions was clear was obtained.

In the present invention, the main material, cellulose acetate, is preferably a mixture of diacetate and triacetate, particularly preferably an equal mixture of both. A porous membrane is made from this by phase separation, which is one of the methods for producing a polymer porous membrane by casting and drying a dope made by dissolving cellulose acetate in its parent solvent, a poor solvent, or a non-solvent in the case of a non-solvent. Done by law.

Examples of the hydrophilic solvent include acetone, ethyl acetate, methylene chloride and the like. Examples of poor solvents are tetrahydrofuran, methanol, ethanol and the like. Water is often used as the non-solvent. 1 to 20 in this mixed solvent
Cellulose acetate is dissolved to a concentration of%.

As a method for adjusting the electroosmotic coefficient of the membrane to less than 0 to minus 3 mm, it is effective to add cellulose ether such as hydroxyethyl, hydroxypropyl or hydroxypropylmethyl, or polyvinyl alcohol as an additive. As the addition method, there is a method such as addition to the dope or a secondary treatment to the formed film. In this case, it is possible to adjust the electroosmosis coefficient to a desired value by adjusting the amount added to the cellulose acetate, and to separate the application point and the fractional zone at an arbitrary distance to some extent.
Therefore, the coating point of this support is 3: 7 to 0: 1 from the negative electrode side.
It can be set in a wide range of 0. However, in practice, from the viewpoint of operation such as application of serum, the range of 3: 7 to 1: 9 is preferable from the negative electrode side, and the position of 2: 8 from the negative electrode side is particularly preferable. If the separation distance between the application point and the fraction zone is less than 1 mm, the accuracy of the measuring instrument such as a densitometer may affect the adjacent γ-globulin fractional value.
It is desirable to separate the above. Also, the coating point is from the negative electrode side 2:
In the case of position 8, in order to develop the protein fraction band in the central part of the support, the electroosmotic coefficient of the membrane must be larger than minus 3.0 mm, preferably minus 1 mm to 2 mm. In this case, the application point and the separation zone are separated by about 2 mm to 3 mm.
The preferable amount of the additive for achieving these purposes is 3 to 5% based on the cellulose acetate as the main component.

Further, as a result of the investigation by the present inventors, it was found that the pore structure of the support greatly affects the migration time, the electric resistance, the sharpness of the fraction band, the presence or absence of the sample residue, and the like. By setting the surface pore size of the support-coated surface of the serum to 10 to 30 μm, preferably 15 to 20 μm, it is possible to obtain a support having no coating residue by preventing adhesion of serum proteins and improving internal permeability of serum. I found it.

However, when the surface pore size and the average pore size are the same, particularly with a support having an average pore size of 10 μm or more, the diffusion phenomenon becomes large during migration and the protein fraction is liable to become unclear. Therefore, by setting the average pore size to 0.2 to 5 μm, preferably 0.4 to 3 μm, and the surface pore size to 10 to 30 μm, preferably 15 to 20 μm, a clear protein fraction band image can be obtained and no coating residue remains at the coating point. It is possible to get a body.

The surface and average pore size of the support can be controlled by the dope composition including the combination of the mixed solvents, the film forming conditions, and the like. Further, in order to improve the wettability of the support, 1 to 15% (by weight) of a nonionic surfactant is added to the cellulose acetate in the dope. Particularly, about 3 to 7% is preferable.
The surface pore size of the support of the present invention is a value obtained by measuring the pore size of the sample-coated surface of the film with an electron microscope. Further, the average pore diameter is represented by the average pore diameter measured by the polysimeter method.

 Hereinafter, the present invention will be described in more detail with reference to experimental examples.

A uniform dope was made with the above composition. This solution was cast on a glass plate to a thickness of 1 mm, allowed to stand for 20 minutes in an atmosphere of a temperature of 25 ° C. and a humidity of 65%, the film produced from the glass plate was peeled off, and further dried in an oven dryer at 60 ° C. It was dried for an hour. The obtained film had the physical properties shown in the above table.

This film is cut into a size of 22 cm × 6 cm, and veronal and veronal sodium buffer (pH 8.6, 0.05 mol /
), And then ADVANTEC Toyosha electrophoresis tank SE
It was loaded in −33 and 0.5 μm each of the germane control serum was applied by an applicator at a length of 1 cm to 20 pieces.
(Coating position: Comparative Example 1, Examples 1 and 2 are from the negative electrode side 2:
8, Comparative Example 2 was applied at a position of 4: 6 from the negative electrode side) and electrophoresed at a current of 0.5 mA / cm for 30 minutes by a conventional method. The membrane was then dissolved in 6% aqueous trichloroacetic acid solution.
It was immersed in a 0.6% Ponceau 3R (Wako Pure Chemical Industries) solution for 3 minutes, dyed, and further decolorized 5 times with a 2% acetic acid aqueous solution for 1 to 2 minutes. As a result, the serum was separated into 5 fractions in Examples 1 and 2 and Comparative Example 1, but was separated into 6 fractions in Comparative Example 2.

 The results are shown below.

One of the commercially available products was electrophoresed under the same conditions as above. The results are as follows.

[Brief description of drawings]

Figures 1 to 4 are electrophoretic image diagrams after staining, decolorization and drying, which were scanned with a densitometer (densitometer: ADC-25EX manufactured by Kagaki Seisakusho), and the absorbance of each fractionation part was continuously measured. First
The figure shows Example 1, FIG. 2 shows Comparative Example 1, FIG. 3 shows Comparative Example 2, and FIG. 4 shows the absorbance of each fractionation part of a commercial product.

Claims (1)

(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 an average pore size of 0.2 to 5 μm and a surface pore size of 10 to 30 μm.