JP2764306B2 - 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 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 are 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.

[Problems of the Related Art] In recent years, a method of reading out 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-A-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. To provide a support for electrophoresis in which an extra peak due to the second fractionation does not occur, and a poor separation property does not occur due to a shift in the position of the β lipoprotein peak due to a difference in β lipoprotein content. is there.

Other objects are alpha ₂ globulin fraction of electrophoresis image density and β-globulin fraction of the electrophoresis image density electrophoresis support the electrophoresis image density gives a high read easily electrophoresis image of significantly reduced contrast between the present invention To provide.

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 electrophoresis support comprising the microporous polymer film described in 262550.

[Means for Solving the Problems] The present invention provides an additive comprising at least one kind of higher fatty acid having 10 to 27 carbon atoms in a weight ratio to a polymer of 10 ppm to 3 ppm.
A support for electrophoresis comprising a polymer microporous film, which is contained in the range of 000 ppm or in the range of 10 ppm to solubility.

[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 by a carbon number of 10 including carbon atoms of a carboxyl group as an additive.
One or more of the higher fatty acids in the range of 27 to about 10 ppm to about 3000 ppm by weight of the polymer (with a solubility of 3000
In the case of fatty acids of less than ppm, it is contained in the range of about 10 ppm to the solubility).

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.

Higher fatty acid in the range of carbon atoms 10 27 including the carbon atom of the carboxyl group used as an additive formula _{C n H 2n + 1 COOH (} n is an integer from 8 to 26) linear represented by Or a branched saturated carboxylic acid. Any of the higher fatty acids represented by the above general formula may be used alone, or two or more arbitrary fatty acids may be used in combination. The content of the higher fatty acid is from about 10 ppm to about 3% by weight relative to the polymer material constituting the polymer microporous film.
000 ppm range (about 10 ppm to solubility for fatty acids with 3000 ppm or less solubility), preferably about 10 ppm to about 1000 ppm (about 10 ppm to solubility for fatty acids with 1000 ppm or less solubility) ).

Specific examples of the higher fatty acids and their preferable addition amounts are as follows.

("C number" is the number of carbon atoms including the carbon atom of the carboxyl group, those without notes are linear fatty acids) Preferred among these higher fatty acids are nonadecanoic acid (nonadecylic acid), eicosanoic acid (arachidic acid), heneicosanoic acid, docosanoic acid (behenic acid), tricosanoic acid, tetracosanoic acid (lignoceric acid), pentacosanoic acid, and hexacosanoic acid ( (Cerotic acid).

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, a polymer material is substantially uniformly dissolved in a solvent, a higher fatty acid, and a plasticizer or a wetting agent component to be added if necessary, in a mixed solvent described later. 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 uniform solution is cast on a temporary support having a smooth surface, dried under a controlled temperature,
By peeling the formed film from the temporary support, a microporous polymer film 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 lyophilic 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, and methanol and ethanol for the polyamide resin. There is.

Specific examples of the poor solvent include, for example, tetrahydrofuran and methanol for cellulose acetate, butanol and ethanol for nitrocellulose, and tetrahydrofuran, dioxane and ethyl acetate for polyamide resin.

 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 microporous polymer film used for the electrophoresis support of the present invention is described in Proc. Nat by EWWash Bahn.
l.Acad.Sci. ”No 1,1115 (1921), Tominaga Keii, Kyoritsu Zensho 157,“ Adsorption ”(p.130), Renichi Kondo,“ Porous Materials ”(Gihodo, 1973),“ Chemical Engineering ” Volume 31, 60-66 (1967)
Etc. are values measured by the mercury penetration method. The pore size distribution ranges from about 0.1 μm to about 10 μm, preferably about 0.1 μm.
Those having a diameter of 97% within the range from μm to about 2.0 μm can be used. Here, the pore size distribution refers to a pore size-cumulative ratio table obtained by the mercury intrusion method, and shows that the cumulative ratio ranges from 3% to 97%.
% Means a range of pore diameters corresponding to the range of%.

Examples 1 to 19 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.
The resulting microporous film was dried at 0 ° C. for 1 hour to prepare a microporous film. 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 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 1.2 g methylene chloride 600 g methanol 280 g water 40 g hydroxypropyl cellulose (hydroxypropyl Higher fatty acid: The following compounds: 10 ppm; 100 ppm; 1000 ppm by weight based on the total amount of cellulose triacetate and cellulose diacetate (“c number” indicates carbon atoms including carboxyl group carbon atoms) Represents a number) [Performance Evaluation Experiment 1] Microporous film 60 prepared in Examples 1 to 19 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 serum coating line (70 mm width direction) along the surface of the film to electrophoretize the serum proteins. After the electrophoresis, the film is stained with Ponceau-3R staining solution. (A dyeing aqueous solution having a composition ratio of 3 g of Ponceau-3R, 6 g of trichloroacetic acid, and 91 g of water) was dyed by immersion for 60 seconds, and washed with a 1% aqueous acetic acid solution 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 five peaks shown in the figure (ALB, α ₁ , α ₂ ,
β and γ).

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

Regarding the peaks α ₂ and β and the minimum value C point, X ₁ = optical density value of α ₂ −optical density value of point C X ₂ = optical density value of β−optical density value of point C, where X ₁ and X ₂ are It is assumed that the larger is the better the separability.

The values of X ₁ and X ₂ for the microporous porous membranes prepared in Examples 1 to 19 and Comparative Example 1 are as follows. From the following data, it is clear that the microporous membrane of the present invention has large values of X ₁ and X ₂ and has good separability. The following data is the distance on the optical density change curve graph of the migration pattern (unit: m
m), not the difference in optical density values. -Indicates that the higher fatty acid is not dissolved.

[Performance evaluation experiment 2] In performance evaluation experiment 1, after electrophoresis, Ponceau-3R
After staining the membrane with the electrophoresis pattern, the membrane was stained by the ozone-Schiff blue staining method for β lipoprotein, and then the distance between the β lipoprotein peak position (β _L ) and the β peak position (β) the (β _L -β) was measured. The following data represents the distance (unit: mm) on the optical density change curve graph of the migration pattern. -Indicates that the higher fatty acid is not dissolved.

In the performance evaluation experiment 1, in the film having large X ₁ and X ₂ , the distance between the β lipoprotein peak position and the β peak position was large, so that interference of the migration pattern due to β lipoprotein was eliminated, and the α ₂ peak position was eliminated. And the β peak position could be clearly distinguished.

Blue staining of β-lipoprotein by the ozonated Schiff method Preparation of Schiff staining solution 13.7 g of sodium sodium sulfite, 21 mL of concentrated hydrochloric acid, fuchsin
8.0 g was dissolved in distilled water to make a total volume of 2000 mL, and stirred overnight with a stirrer. Next, 5 tablespoons of activated carbon were added to this solution, and the insoluble components were filtered with filter paper over 15 minutes, and the filtered solution was refrigerated at about 4 ° C.

Preparation method of Schiff washing solution 4.3 g of sodium pyrosulfite and 9 mL of concentrated hydrochloric acid in 887 g of distilled water
And 100 mL of 95% ethanol was added.

Dyeing operation In the performance evaluation experiment 1, the film dyed with Ponceau-3R and measured for the electrophoretic turn was ozone-oxidized for 20 minutes by an ozone generator (Ozone Japan, Tokyo).

After the ozone-oxidized film was immersed in a Schiff cleaning solution for several seconds, the film was dyed with a Schiff dyeing solution for 10 minutes, and then washed three times with a Schiff cleaning solution for 10 minutes each, and the film was dried with filter paper.

[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 19 (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.

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

Claims (2)

(57) [Claims] 1. An additive comprising at least one higher fatty acid having 10 to 27 carbon atoms in a weight ratio to a polymer.
An electrophoresis support comprising a polymer microporous film, wherein the support is contained in the range of 10 ppm to 3000 ppm or in the range of 10 ppm to solubility. 2. The support for electrophoresis according to claim 1, wherein the polymer constituting the polymer microporous film is at least one polymer selected from the group consisting of cellulose ester, polyamide, and polyvinyl chloride. body.