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A technique for microimmunoelectrophoresis in acrylamide gels
Immunochemistry. Pergamon Press 1968. Vol. 5, pp. 497-499. Printedin Great Britain
A TECHNIQUE FOR MICROIMMUNOELECTROPHORESIS IN ACRYLAMIDE GELS* DIANNA E. VAN ORDEN
Dept. of Obstetrics and Gynecology, University of Iowa, Iowa City, Iowa 52240
(Received 15 February 1968) A PROCEDURE for conducting immunoelectrophoresis in acrylamide gels was described by Antoine in 1962 [1], yet the medium is still rarely used for immunologic reactions. The gel is superior to agar in its physical characteristics and immunoelectrophoretic patterns, but its preparation has several features which may have delayed its general application. The gel cannot be cut, but must be cast under templates to form the wells and troughs. At the present time, these templates must be fabricated on a custom basis, and therefore are quite expensive. In addition, if the riboflavin photocatalysis described by Antoine is to be used, the polymerization must be carried out in an atmosphere of COy This is a minor inconvenience, but adds to the cost of setting up the method in the laboratory. The following report describes a method for molding inexpensive templates in the laboratory, and presents a simplified chemical polymerization procedure adapted from Davis' disc electrophoresis method [2]. Reusable templates having a variety of patterns may be cast of dental acrylic resin as follows: 4% agar in water is poured to a depth of 2-3 m m on a large, siliconized glass plate. After the agar has gelled, it is removed from the plate and laid upside down on glass slides that are the size needed for the final acrylamide pattern. Without touching the surface with the fingers, the agar is trimmed to fit the slide. An additional 2 m m wide strip of agar is removed from each long side so that the resulting acrylic template will have a 'shoulder' along each long edge. After the agar is cut in the desired pattern,~" it is allowed to dry in air for a few minutes to reduce the surface moisture. T o prevent the acrylic polymer from running off the slide during the casting, bars of laundry soap are placed along each side of the slide and are held firmly against the slide edges by a rubber band encircling the two bars of soap and the slide (Fig. 1 a, b). The dental acrylic resin is poured in two layers to avoid the surface irregularities which are produced by the heat liberated during the polymerization of a single, thick layer. A thin mixture of dental acrylic resin + is prepared for the first layer by mixing equal weights of powder and solution. The polymer is flowed into the wells and troughs first, then it is spread over the agar to a uniform thickness of * Supported by Grants from the Population Council and College of Medicine University of Iowa. t Since there is no electroosmotic flow in acrylamide gels, the wells should be cut at a point even with the end of the trough. 'Duz All' Self curing acrylic, Coralite Dental Products, 531 South Plymouth Court, Chicago, Illinois, 60605. 497
DIANNA E. VAN ORDEN
498
b
a
d
FIO. I. (a) Top view of components used in casting templates of dental acrylic resin. Agar (white) on microscope slide. Exposed portion of slide (black). Bars of soap (mottled) placed along each side of slide to prevent resin from running off the slide. (b) End view of slide with bars of soap on each side. (c) End view, showing first layer of dental acrylic resin (checkered) filling the trough and covering the agar surface only. (d) End view, showing second layer of dental acrylic resin (hatched) in place. about 1 m m (Fig. 1 c). After 15-20 min, it is reinforced with a second, more viscous acrylic mixture that is prepared by mixing 5 parts of powder with 3 parts of solution. The second layer should be spread over the whole area between the bars of soap, and should extend the length of the agar (Fig. 1 d). A depth of 2-3 m m over the central portion is adequate. After approximately ½ hr, the bars of soap and the agar form can be removed. If there are burrs or excess acrylic at the ends of the slide, they should be removed with a file or sandpaper. T o mold the acrylamide immunoelectrophoresis slides, the template is laid, pattern side up, in a pan made by folding up the edges of a heavy piece of aluminum foil. Sufficient polymer solution (see below) is added to fill the template and the ends of the foil boat. A clean microscope slide is laid on the 'shoulders' of the template and the whole assembly is placed in an oven at 56 ° for 20 min. After the gel is polymerized the template and slide are rinsed under cool water to remove the excess gel and polymer solution. The template is removed by inserting a spatula under one corner to allow air to enter between it and the gel, which is left undisturbed on the slide. T h e gel can be used immediately, or it can be kept in a moist chamber for several days. Once it is allowed to air dry even slightly, it must be touched only by moist fingers or instruments since it becomes sticky. If this precaution is observed, the gel is easier to handle than agar. The polymer solution is mixed from the following stock solutions: (A) 1 N HCI, 48 ml; tris, 36.6 g; T E M E D , 0.23 ml; water to 100 ml [2]. (B) acrylamide, 9.5 g; N,N'-methylenebisacrylamide, 0.5 g; water to 100 ml. (C) ammonium persulfate, 0.14 g; water to 100 ml. Stock solutions A and B can be kept for several months at refrigerator temperature and warmed prior to use. A fresh solution of C should be
co
FIO. 2. Immunoelectrophoretic pattern of normal human serum developed with horse anti-human serum (Hyland). Unstained acrylamide slide photographed at 72 hr by indirect light.
A Technique for Microimmunoelectrophoresis in Acrylamide Gels
499
made each week. The polymer solution is prepared by mixing one part A, three parts B and four parts C just prior to filling the templates. Exceptionally good patterns are obtained when the electrophoresis is carried out with an ethylenediamine-acetic acid tank buffer [3]. Electrophoresis may be performed at room temperature with the current maintained at 4 mA/cm of slide width until a bromphenol blue-albumin marker has migrated to within 1 cm of the end of the trough (1-2 hr). When diffusion is allowed to take place at room temperature, the arcs begin to appear within 12 hr after the addition of the antiserum, and are generally complete at 48-72 hr. The gel is remarkably resistant to bacterial or fungal growth; an acrylamide slide remains uncontaminated even if kept for a week in a moist chamber with a contaminated agar slide. This resistance along with the fine resolution of even minor arcs makes acrylamide a good medium for the study of weak antigens. Photography is the easiest method for recording the immunoelectrophoretic patterns since the fine arcs show up well against the water-clear gel (Fig. 2). Lint and accumulated dust should be washed from the gel surface and the troughs and wells filled with water to eliminate reflections from the edges. The best reproduction is obtained with dark field illumination and a film of moderate speed and contrast such as Kodak Panatomic-X. If it is deemed necessary to stain the slides, they must be soaked free of unreacted proteins over a period of several days. The gels can be stained with a variety of general and specific stains without first drying the gel [4]. Amidoschwartz is a sarisfactory protein stain. It can be destained with 7% acetic acid by leaching or by electrophoresis if an appropriate migration chamber is available. The periodic acidSchiff reaction is excellent for carbohydrates since there is no need to destain the background. For the same reason 2% osmium tetroxide would be advantagous for staining lipids. The stained slides can be air-dried if it is done slowly. If the drying is done too rapidly, the gel becomes brittle and cracks. The dried, stained, slides can be preserved by spraying them with a clear plastic spray.
1. 2. 3. 4.
REFERENCES ANTOI~CEB., Science 135, 977 (1962). DAvis B.J., Ann. N.Y. Acad. Sd. 1219 404 (1964). CROWLEA. J., Immgnodiffusion p. 303. Academic Press, New York (1961). CLARKEJ.T., Ann. N.Y. Acad. Sci. 121~ 428 (1964).
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A TECHNIQUE FOR MICROIMMUNOELECTROPHORESIS IN ACRYLAMIDE GELS* DIANNA E. VAN ORDEN
Dept. of Obstetrics and Gynecology, University of Iowa, Iowa City, Iowa 52240
(Received 15 February 1968) A PROCEDURE for conducting immunoelectrophoresis in acrylamide gels was described by Antoine in 1962 [1], yet the medium is still rarely used for immunologic reactions. The gel is superior to agar in its physical characteristics and immunoelectrophoretic patterns, but its preparation has several features which may have delayed its general application. The gel cannot be cut, but must be cast under templates to form the wells and troughs. At the present time, these templates must be fabricated on a custom basis, and therefore are quite expensive. In addition, if the riboflavin photocatalysis described by Antoine is to be used, the polymerization must be carried out in an atmosphere of COy This is a minor inconvenience, but adds to the cost of setting up the method in the laboratory. The following report describes a method for molding inexpensive templates in the laboratory, and presents a simplified chemical polymerization procedure adapted from Davis' disc electrophoresis method [2]. Reusable templates having a variety of patterns may be cast of dental acrylic resin as follows: 4% agar in water is poured to a depth of 2-3 m m on a large, siliconized glass plate. After the agar has gelled, it is removed from the plate and laid upside down on glass slides that are the size needed for the final acrylamide pattern. Without touching the surface with the fingers, the agar is trimmed to fit the slide. An additional 2 m m wide strip of agar is removed from each long side so that the resulting acrylic template will have a 'shoulder' along each long edge. After the agar is cut in the desired pattern,~" it is allowed to dry in air for a few minutes to reduce the surface moisture. T o prevent the acrylic polymer from running off the slide during the casting, bars of laundry soap are placed along each side of the slide and are held firmly against the slide edges by a rubber band encircling the two bars of soap and the slide (Fig. 1 a, b). The dental acrylic resin is poured in two layers to avoid the surface irregularities which are produced by the heat liberated during the polymerization of a single, thick layer. A thin mixture of dental acrylic resin + is prepared for the first layer by mixing equal weights of powder and solution. The polymer is flowed into the wells and troughs first, then it is spread over the agar to a uniform thickness of * Supported by Grants from the Population Council and College of Medicine University of Iowa. t Since there is no electroosmotic flow in acrylamide gels, the wells should be cut at a point even with the end of the trough. 'Duz All' Self curing acrylic, Coralite Dental Products, 531 South Plymouth Court, Chicago, Illinois, 60605. 497
DIANNA E. VAN ORDEN
498
b
a
d
FIO. I. (a) Top view of components used in casting templates of dental acrylic resin. Agar (white) on microscope slide. Exposed portion of slide (black). Bars of soap (mottled) placed along each side of slide to prevent resin from running off the slide. (b) End view of slide with bars of soap on each side. (c) End view, showing first layer of dental acrylic resin (checkered) filling the trough and covering the agar surface only. (d) End view, showing second layer of dental acrylic resin (hatched) in place. about 1 m m (Fig. 1 c). After 15-20 min, it is reinforced with a second, more viscous acrylic mixture that is prepared by mixing 5 parts of powder with 3 parts of solution. The second layer should be spread over the whole area between the bars of soap, and should extend the length of the agar (Fig. 1 d). A depth of 2-3 m m over the central portion is adequate. After approximately ½ hr, the bars of soap and the agar form can be removed. If there are burrs or excess acrylic at the ends of the slide, they should be removed with a file or sandpaper. T o mold the acrylamide immunoelectrophoresis slides, the template is laid, pattern side up, in a pan made by folding up the edges of a heavy piece of aluminum foil. Sufficient polymer solution (see below) is added to fill the template and the ends of the foil boat. A clean microscope slide is laid on the 'shoulders' of the template and the whole assembly is placed in an oven at 56 ° for 20 min. After the gel is polymerized the template and slide are rinsed under cool water to remove the excess gel and polymer solution. The template is removed by inserting a spatula under one corner to allow air to enter between it and the gel, which is left undisturbed on the slide. T h e gel can be used immediately, or it can be kept in a moist chamber for several days. Once it is allowed to air dry even slightly, it must be touched only by moist fingers or instruments since it becomes sticky. If this precaution is observed, the gel is easier to handle than agar. The polymer solution is mixed from the following stock solutions: (A) 1 N HCI, 48 ml; tris, 36.6 g; T E M E D , 0.23 ml; water to 100 ml [2]. (B) acrylamide, 9.5 g; N,N'-methylenebisacrylamide, 0.5 g; water to 100 ml. (C) ammonium persulfate, 0.14 g; water to 100 ml. Stock solutions A and B can be kept for several months at refrigerator temperature and warmed prior to use. A fresh solution of C should be
co
FIO. 2. Immunoelectrophoretic pattern of normal human serum developed with horse anti-human serum (Hyland). Unstained acrylamide slide photographed at 72 hr by indirect light.
A Technique for Microimmunoelectrophoresis in Acrylamide Gels
499
made each week. The polymer solution is prepared by mixing one part A, three parts B and four parts C just prior to filling the templates. Exceptionally good patterns are obtained when the electrophoresis is carried out with an ethylenediamine-acetic acid tank buffer [3]. Electrophoresis may be performed at room temperature with the current maintained at 4 mA/cm of slide width until a bromphenol blue-albumin marker has migrated to within 1 cm of the end of the trough (1-2 hr). When diffusion is allowed to take place at room temperature, the arcs begin to appear within 12 hr after the addition of the antiserum, and are generally complete at 48-72 hr. The gel is remarkably resistant to bacterial or fungal growth; an acrylamide slide remains uncontaminated even if kept for a week in a moist chamber with a contaminated agar slide. This resistance along with the fine resolution of even minor arcs makes acrylamide a good medium for the study of weak antigens. Photography is the easiest method for recording the immunoelectrophoretic patterns since the fine arcs show up well against the water-clear gel (Fig. 2). Lint and accumulated dust should be washed from the gel surface and the troughs and wells filled with water to eliminate reflections from the edges. The best reproduction is obtained with dark field illumination and a film of moderate speed and contrast such as Kodak Panatomic-X. If it is deemed necessary to stain the slides, they must be soaked free of unreacted proteins over a period of several days. The gels can be stained with a variety of general and specific stains without first drying the gel [4]. Amidoschwartz is a sarisfactory protein stain. It can be destained with 7% acetic acid by leaching or by electrophoresis if an appropriate migration chamber is available. The periodic acidSchiff reaction is excellent for carbohydrates since there is no need to destain the background. For the same reason 2% osmium tetroxide would be advantagous for staining lipids. The stained slides can be air-dried if it is done slowly. If the drying is done too rapidly, the gel becomes brittle and cracks. The dried, stained, slides can be preserved by spraying them with a clear plastic spray.
1. 2. 3. 4.
REFERENCES ANTOI~CEB., Science 135, 977 (1962). DAvis B.J., Ann. N.Y. Acad. Sd. 1219 404 (1964). CROWLEA. J., Immgnodiffusion p. 303. Academic Press, New York (1961). CLARKEJ.T., Ann. N.Y. Acad. Sci. 121~ 428 (1964).
i~uNo. 5/5--G--z