JP2802645B2 - Electrophoresis support - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a support for electrophoresis comprising a microporous polymer film, and more particularly to a support suitable for fractionating serum proteins. It is.

[Prior Art] Electrophoresis is used for separating and purifying charged substances, and is particularly useful for separating and fractionating proteins. For electrophoresis, in addition to general electrophoresis, a discontinuous buffer system is used, disc electrophoresis for separation, immunoelectrophoresis for separation and detection by immunodiffusion, and pH gradient There is a method such as an isoelectric point fractionation method in which the particles are formed in the middle and separated and fractionated in the pH gradient.

Fractionation of serum proteins is widely performed in the field of clinical testing, and for this purpose, a large number of electrophoresis supports composed of a microporous film mainly composed of a polymer such as cellulose acetate have been used. I have.

As a method for fractionating serum proteins by electrophoresis using a cellulose acetate microporous membrane as a polymer microporous support, immersing the microporous membrane in a buffer solution with a concentration of 0.03 to 0.08 mol / L and pH 8.6 Then, serum is applied to the surface of the microporous membrane, a direct current is applied, the protein is electrophoresed, fractionated, and then the protein is stained. As the dye for dyeing, dyes such as Ponceau-3R, Ponceau-S, nigrosine, amide black, Coomassie brilliant blue and the like are used. Thus fractionation dye images obtained are usually divided into five fractions, albumin fraction in order from the anode side, alpha ₁ globulin fraction, alpha ₂ globulin, beta globulin fraction, gamma
It is named the globulin fraction.

Serum proteins are roughly divided into five fractions on electrophoretic analysis. However, each of the five fractions does not belong to the same chemical component and happens to have mobility due to electricity. In this case, components belonging to completely different types in chemical structure only coexist in one fraction. For example, in the β globulin fraction, in addition to the globulin component, a complex of LDL cholesterol and a protein coexists. Also the alpha ₂ globulin fraction, in addition to the globulin components include complexes of VLDL cholesterol and proteins.

Fractionation of serum proteins by electrophoresis
In terms of chemical structure, it is not a simple and straightforward classification into five strains, but as a primary screening of clinical tests, it is first divided into five fractions, and each fraction value is significantly different from the normal value. This is done to confirm that they are not present. As a primary screening, the above-mentioned five fractions are first tested, and if a problematic result is obtained, a more precise protein test is usually performed.

[Related Art and Problems] In recent years, a method of reading a test result by digital or analog output from a computer by an automatic analyzer has become widespread. While the automatic reading method has the advantage that the disease state can be detected more accurately, the serum protein is usually used for 5 times.
During classification, if small fluctuations cause extra peaks or irregular peak intervals, confusing pattern recognition by automatic processing may result in erroneous reading. . Therefore, even if a slight variation or irregularity of peak intervals occurs, in the serum fractionation for the purpose of the primary screening, the support for electrophoresis has a fractionation property such that almost all sera can be distinguished into 5 fractions. The development of is desired.

In the case of VLDL cholesterol or a complex of LDL cholesterol and a protein (referred to as pre-β lipoprotein and β-lipoprotein, respectively), the fractionated position differs depending on the content. For example, alpha ₂ globulin fraction (third fraction) beta
A β-lipoprotein fraction (sixth fraction) is generated between the globulin fraction (fourth fraction), and this β-lipoprotein fraction is
Depending on the content of β lipoprotein alpha ₂ globulin fraction (Third
Fraction) and β-globulin fraction (fourth fraction) is located between are included in the alpha ₂ globulin fraction is (the content is less variation, if the content is increased alpha ₂ globulin fraction and β-globin Independent fractionation between fractions). Thus the sixth
When the second fraction appears, it often becomes an obstacle to judge the test result based on five fractions.

This obstacle is, for example, a support for electrophoresis comprising a microporous polymer film having a pore size distribution range of 0.1 μm to 2.0 μm described in JP-B-63-262549, a specific wetting agent described in JP-A-63-262550. Alternatively, it was improved by an electrophoresis support comprising a microporous polymer film to which a plasticizer was added at a ratio of 20% or less based on the weight of the polymer, but the situation was not yet solved. Furthermore, even with these electrophoretic supports, the irregularity of the peak intervals of the proteins separated still remained unresolved.

[Problems to be Solved by the Invention] An object of the present invention is to provide a support for electrophoresis which can be widely applied to various kinds of serums. An object of the present invention is to provide a support for electrophoresis, in which an extra peak due to the second fractionation does not occur and the intervals between the peaks of the separated proteins do not become uneven.

Another object of the present invention is to provide a substantially dry support for electrophoresis which is easy to handle.

Other objects of the present invention are described in JP-A-63-262549 and JP-A-63-262549.
An object of the present invention is to provide an improved electrophoretic support comprising the microporous polymer film described in 262550.

[Means for Solving the Problems] The present invention relates to a support for electrophoresis comprising a polymer microporous film comprising hydroxypropyl cellulose, triphenyl phosphate or tricresyl phosphate as an additive. It is.

[Detailed Description of the Structure of the Invention] The electrophoresis support comprising the polymer microporous film of the present invention is disclosed in JP-B-55-31418, JP-A-63-262549 and JP-A-63-262549.
An improved electrophoretic support comprising a microporous polymer film described in 3-262550, characterized in that it contains hydroxypropyl cellulose, triphenyl phosphate or tricresyl phosphate as an additive. .

The support for electrophoresis comprising the polymer microporous film of the present invention is a substantially dry film, having a thickness of about 50 μm to about 300 μm.
μm, preferably from about 100 μm to about 200 μm, most preferably from about 100 μm to about 150 μm. The pore size of the pores ranges from about 0.1 μm to about 10 μm, preferably from about 0.1 μm to about 2.0 μm. The porosity of the pores is about 50
% To about 90%, preferably about 60% to about 85%.

The support for electrophoresis comprising the polymer microporous film of the present invention is disclosed in JP-B-55-31418, JP-A-50-12256, and JP-A-55-
76360, JP-A-63-262549, JP-A-63-262550 and the like.

Nitrocellulose, cellulose acetate (cellulose diacetate, cellulose triacetate, etc.), cellulose ester such as cellulose acetate butyrate, cellulose propionate, polyamide resin, polyvinyl chloride resin Etc. The polymer materials can be used alone or in combination of two or more. Among these polymer materials, cellulose esters and polyamide resins are preferred, and cellulose acetate (cellulose diacetate, cellulose triacetate) is most preferred.

As the hydroxypropyl cellulose used as an additive, the content of the hydroxypropyl group is from about 55% to about 55%.
Some are in the 75% range. Any one of hydroxypropyl cellulose, triphenyl phosphate and tricresyl phosphate may be used, or any two or three of these three may be used in combination. The content of hydroxypropylcellulose, triphenyl phosphate or tricresyl phosphate is about 0.1% by weight with respect to the polymer material constituting the polymer microporous film.
To about 1.0%, preferably from about 0.2% to about 0.6%.

The polymer microporous film may contain a known plasticizer or wetting agent. The content of the plasticizer or wetting agent is in the range of about 1% to about 20% by weight with respect to the polymer material in order to make the microporous film flexible. Specific examples of useful plasticizers or wetting agents include diols such as ethylene glycol, diethylene glycol, propylene glycol, tetramethylene glycol, glycerol monoacetyl ester, glycerol, triethyl citrate, tributyl citrate, trimethyl citrate. , Diethyl oxalate, dipropyl oxalate, tributyrin (glycerol tributyrate), triacetin (glycerol triacetate), diethyl succinate, dibutyl succinate and the like.

A method for producing the microporous polymer film will be described. First, the polymer material is substantially uniformly dissolved in a solvent and an additive (hydroxypropylcellulose, triphenyl phosphate or tricresyl phosphate), and a plasticizer or a wetting agent component which is optionally added is mixed in a mixed solvent described below. . The resulting solution is used to prepare a microporous film according to a known method described in the above-mentioned patent specifications and the like. For example, a microporous polymer film is formed by casting a uniform solution on a temporary support having a smooth surface, drying it under a controlled temperature, and peeling the formed film from the temporary support. Is obtained.

The mixed solvent refers to a mixed solvent obtained by mixing the three components of a lipophilic solvent, a poor solvent and a non-solvent. Here, the lipophilic solvent means a substance that dissolves the polymer material, and the poor solvent is compatible with the lipophilic solvent, but does not substantially dissolve the polymer material, only swells, and more than the lipophilic solvent. A non-solvent is one that is compatible with a lyophilic solvent or a poor solvent, but does not dissolve or swell the polymer material and has a higher boiling point than the lipophilic solvent.

Specific examples of the lipophilic solvent include, for example, methylene chloride, acetone, methyl formate and the like for cellulose acetate, diethyl ether, methyl acetate, acetone, acetic acid and the like for nitrocellulose, methanol for polyamide resin, There are ethanol and the like.

To give specific examples of poor solvents, for example, for cellulose acetate, tetrahydrofuran, methanol and the like,
For nitrocellulose, butanol, ethanol and the like, and for polyamide resin, there are tetrahydrofuran, dioxane, ethyl acetate and the like.

 Water is often used as the non-solvent.

The distinction between the lipophilic, poor and non-solvent of these solvents is not always unique. The above-described production method is an example for obtaining the support for electrophoresis of the present invention.

With respect to the use of the solvent, especially the use of the lipophilic solvent and the poor solvent, JP-B-55-31418, JP-A-50-122565, JP-A-51-763.
The method described in the specification such as 60 can be applied.

The pore size distribution range of the polymer microporous coating used for the electrophoresis support of the present invention is described in Proc. Nat by EWWash Bahn.
l.Acad.Sci. "(1), 1115 (1921), Tominaga Keii, Kyoritsu Zensho 157" Adsorption "(p. 130), Renichi Kondo," Porous Materials "
(Gihodo, 1973), “Chemical Industry”, Vol. 31, pp. 60-66 (1967)
Year) etc. and measured by the mercury thickening method. A pore size distribution range of about 0.1 μm to about 10 μm, preferably a range of about 0.1 μm to about 2.0 μm with a diameter of 97% can be used. Here, the pore diameter distribution means a pore diameter range corresponding to a cumulative rate in the range of 3% to 97% according to a pore diameter-cumulative rate table obtained by a mercury intrusion method.

Examples 1 to 3 and Comparative Example 1 [Preparation of Microporous Film] A substantially uniform solution having the following composition was prepared, and each solution was placed on a glass plate (temporary support) having a smooth surface. 1mm thick
And dried at 25 ° C. When the entire surface of the film becomes almost uniformly white, peel it off from the glass plate and put it on a frame for another 10 minutes.
After drying at 0 ° C. for 1 hour, a microporous film was prepared. Each of the obtained microporous films had a dry thickness of about 150 μm and a pore size range of 0.1 μm to 2.0 μm.

Solution composition (* indicates the amount shown in Table 1) Cellulose triacetate (acetyl group content 43.5
%) 30 g Cellulose diacetate (acetyl group content 39.4%) 30 g glycerol 4.2 g tributyrin 4.8 g triphenyl phosphate * methylene chloride 600 g methanol 280 g water 40 g hydroxypropyl cellulose (hydroxypropyl group content 64.0%) * methyl cellulose (containing methoxy group) 29.8%) * [Performance Evaluation Test] Microporous film 4 prepared in Examples 1 to 3 and Comparative Example 1
Seeds are cut to a size of 70 mm x 200 mm, each having a pH of 8.6
0.07M veronal pH buffer aqueous solution (2.30 g of veronal and 1
Dissolve 1.80 g of veronal sodium in distilled water, make the total volume 1000 mL at 25 ° C), set these films in an electrophoresis tank filled with the same buffer, and place the filter paper as a bridge. did. With a micropipette, linearly
Human serum was applied at a distance of 15 mm from the cathode end of the microporous film in a total volume of 0.1 μL over 10 mm. A constant DC current of 0.6 mA / cm is applied for 40 minutes in the direction perpendicular to the line of serum application (70 mm width direction) along the surface of the membrane, and the serum proteins are electrophoresed. After electrophoresis, the membrane is stained with Ponceau-3R staining solution (Dyeing aqueous solution having a composition ratio of 3 g of Ponceau-3R, g of trichloroacetic acid, and 91 g of water) was dyed by dipping for 60 seconds, and washed with a 1% aqueous solution of acetic acid four times for 2 minutes each.

After washing, filter paper was applied to both sides of the film, the cleaning solution was sucked out, and the film was air-dried.

After air drying, the serum protein migration pattern was observed.
The number of protein fractions in all microporous polymer membranes was 5
Thus, the sixth fraction by β-lipoprotein did not appear. When the change in optical density of the stained electrophoresis pattern was measured by a densitometer, the electrophoresis pattern showed five peaks (ALB, α ₁ , α ₂ ,
β and γ).

Regarding the optical density change curve of the migration pattern shown in FIG. 1, the resolution is defined as follows.

The peak intervals of ALB, α ₁ , α ₂ , and β are represented by relative ratios. A
The relative ratio of the intervals of LB, α ₁ , α ₂ , and β is 1: 1: 1
It is assumed that the separation performance is good when is close to.

The separation performance improved in the support for electrophoresis of the present invention is a balance between the peak intervals of ALB, α ₁ , α ₂ , and β, which is expressed by a relative ratio of each peak interval and evaluated by comparing the balance. ALB-alpha ₁ for microporous membranes of examples 1 to 3 and Comparative example 1 in table 2 which can _be, alpha _{1-.alpha. 2,} shows the relative ratio of the alpha _2-beta respective peak intervals. The numerical value in parentheses indicates a relative value of the peak interval when the minimum value of the peak interval is set as the reference value (1.00).

Comparative Example 1 in which the microporous porous membranes (Examples 1 to 3) according to the present invention are the prior art from the values of the relative ratios of the peak intervals in Table 2
ALB-α ₁ , α ₁ -α2, α
The relative ratio of the peak spacing of each of the _2- β is close to 1: 1: 1,
It can be seen that the separation performance is good.

[Brief description of the drawings]

FIG. 1 shows the change in optical density of the migration pattern obtained by electrophoresing and staining human serum using the microporous film according to Examples 1 to 3 (invention) and Comparative Example 1 (prior art). It is a schematic diagram of the optical density change curve measured by the meter. The curve shows serum protein components ALB, α ₁ , α ₂ , β, γ
And their intervals.

Continuation of front page (56) References JP-A-63-262550 (JP, A) JP-A-63-262549 (JP, A) JP-A-50-122565 (JP, A) JP-B-55-31418 (JP, A) , B2) (58) Field surveyed (Int. Cl. ⁶ , DB name) G01N 27/447

Claims (2)

(57) [Claims] 1. A support for electrophoresis comprising a polymer microporous film characterized by containing hydroxypropyl cellulose, triphenyl phosphate or tricresyl phosphate as an additive. 2. The polymer constituting the polymer microporous film is at least one polymer selected from the group consisting of cellulose ester, polyamide, and polyvinyl chloride, and is embodied in the polymer of the additive. Content from 0.1% by weight
2. The support for electrophoresis according to claim 1, which is in a range of 1.0%.