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Immunoelectrophoresis 1 MBTCC-6;/M.SC. (BIO.); UNIT V; IMMUNOELECTROPHORESIS Immunoelectrophoresis is a general name for separation and characterization of proteins based on electrophoresis and reaction with antibodies. All variants of immunoelectrophoresis require immunoglobulins, also known as antibodies reacting with the proteins to be separated or characterized. Some antigen mixtures are too complex to be resolved by simple diffusion and precipitation. Greater resolution is obtained by the technique of classical immunoelectrophoresis, in which antigens are first separated based on their electrical charge, then visualized by the precipitation reaction. In this procedure antigens are separated by electrophoresis in an agar gel. Positively charged proteins move to the negative electrode, and negatively charged proteins move to the positive electrode (figure 33.16a). A trough is then cut next to the wells (figure 33.16b) and filled with antibody. If the plate is incubated, the antibodies and antigens will diffuse and eventually form precipitation bands or arcs (figure 33.16c) that can be better visualized by staining (figure 33.16d). This assay is used to separate the major blood proteins in serum for certain diagnostic tests. In immunoelectrophoresis, the proteins are first separated by horizontal agarose gel electrophoresis on the basis of their different charge-to-mass ratios. The gel with the separated proteins (antigens) is then removed from the electric field and antibodies to the proteins are introduced into narrow troughs parallel to the separated antigens. Diffusion of both antigen antibody takes place and, at a particular locus, the equivalence point is reached resulting in precipitation. Procedure: 1. Prepare the agarose gel solution 2. Before pouring the gel solution into the casting tray, construct and place (in the middle of the tray) a trough former using two microscope slides and the bottom half of a small (60 mm) petri plate. Tape one microscope slide to the bottom of the petri plate (see Figure 1). Again, Tape the second microscope slide to the opposite side of the petri plate so it is even with the first slide. From the side view, the slides should be even and parallel. Figure 1 Dr. V. K. Mishra, University Department of Biotechnology, L. N. Mithila University, Kameshwarnagar, Darbhanga 2 3. Before the molten agarose solidifies, place the trough former in the middle of the tray to simultaneously form the two troughs. See Template on Figure 2. 4. Prepare a humidifying chamber and Add DDW to saturate the towels and then Cover it with lid, plastic wrap, or foil. 5. Prepare electrophoresis wicks by cutting filter paper into strips approximately 7 cm x 3 cm. The ends of the wicks must be long enough to extend into electrophoresis buffer in the chamber (see Figure 2). Figure 2 ELECTROPHORETIC SEPARATION OF ANTIGENS 1. Cut wells where indicated in Figure 2 using well cutter/gel punch. The distance between the troughs and the edge of each well should not be more than 0.5 cm. 2. Remove agarose gel plugs with a toothpick or spatula. 3. Transfer the tray containing the gel to the electrophoresis apparatus. 4. Gently lay the filter paper wicks over the ends of the gel, (They should overlap about 3 to 4 mm) and allow them to become saturated with electrophoresis buffer. 5. The wicks should be submerged in the buffer. Press lightly on the wicks to ensure good contact between the gel and the electrophoresis buffer. If necessary, add more buffer, but do not cover the gel with buffer. 6. Load 20 μl of each antigen (A, B, and C) in the wells as indicated by the diagram 7. Turn on and set the power supply for the required voltage (50V to 125V). When current is flowing properly, bubbles should form on the electrodes. 8. Run the electrophoresis until the blue dye has migrated to the ends of the troughs. The exact time required is dependent upon the voltage NOTE: The samples contain dye which will migrate at different rates. Terminate electrophoresis when the first dye reaches just past the end of the troughs. Do not allow the samples to migrate off the end of the gel. 9. After electrophoresis is completed, turn off the power, unplug the power source, disconnect the leads, and remove the cover. 10. Discard the filter paper wicks and remove the gel tray from the apparatus. Set the tray on a level surface and proceed with the diffusion steps. NOTE: Do not add the antibody samples in the troughs until after electrophoretic separation. DIFFUSION OF ANTIBODIES AND ANTIGENS 1. Add 50 μl of each antibody to the appropriate trough (see Figure 2). Use the pipette tip to carefully spread the antibody solution along the entire length of the trough. 2. Place the tray in a closed humidifying chamber containing moistened paper towels. Dr. V. K. Mishra, University Department of Biotechnology, L. N. Mithila University, Kameshwarnagar, Darbhanga 3 3. Allow diffusion to take place over a 24 to 48 hour period, or until visible precipitates form in the gel. The chamber can be placed in a 37°C incubation oven or remain at room temperature. RESULTS 1. Note the formation of arcs of white precipitate in the gel. 2. Identify the number of proteins in the whole serum from the number of arcs of precipitate. Four to six arcs can be expected, corresponding to various albumins found in Whole Serum. 3. Identify albumin and IgG in the whole rabbit serum from comparison with the pure albumin and the pure anti-IgG segments. The prominent precipitant arc corresponds to the complex formed by the reaction of the anti-IgG with pure IgG. Applications: In the clinical laboratory, immunoelectrophoresis is used diagnostically. It is utilized in examining certain serum abnormalities, especially those involving immunoglobulins, urine protein, cerebrospinal fluid, pleural fluids and other body fluids. In research, this procedure may be used to monitor antigen and/or antibody purifications, to detect impurities, analyze soluble antigens from plant and animal tissues, and microbial extracts. Figure 3 Rocket Immunoelectrophoresis Rocket Immunoelectrophoresis is an adaptation of radial immunodiffusion developed by Laurell. It is called as “rocket electrophoresis” due to the appearance of the precipitin bands in the shape of cone-like structures (rocket appearance) at the end of the reaction. In this technique antigen migrates in an electric field in a layer of agarose containing an appropriate antibody. The migration of the antigen toward the anode gives rise to rocket-shaped patterns of precipitation. The area under the rocket is proportional to antigen concentration. This technique is used to detect antigen-antibody complexes. And to Determine the concentration of antigen in an unknown sample. PRINCIPLE This is a quantitative one-dimensional single electro-immunodiffusion technique. In this method antibody is incorporated in the gel at a pH value at which the antibodies remain Dr. V. K. Mishra, University Department of Biotechnology, L. N. Mithila University, Kameshwarnagar, Darbhanga 4 essentially immobile. Antigen is placed in wells cut in the gel. Electric current is then passed through the gel, which facilitates the migration of negatively charged antigens into the agar. As the antigen moves out of the well and enters the agarose gel, it combines with the antibody to form immune complex which becomes visible. During the initial phase there is considerable antigen excess over antibody and no visible precipitation occurs. However, as the antigen sample migrates further through the agarose gel, more antibody molecules are encountered that interact with the antigen to form immune complex. This results in formation of a precipitin line that is conical in shape, resembling a rocket. The greater the amount of antigen loaded in a well, the further the antigen will have to travel through the gel before it can interact with sufficient antibody to form a precipitate. Thus, the height of the rocket, measured from the well to the apex and area are directly proportional to the amount of antigen in the sample. PROCEDURE 1. Prepare 15 ml of 1 % agarose gel. 2. Cool down the solution to 55-60oC and add 250 ml of antiserum to 13 ml of agarose solution. Mix homogenously for uniform distribution of antibody. 3. Pour agarose solution containing the antiserum on to grease free glass plate placed on a horizontal surface and allowed the gel to set for 30 minutes. 4. The glass plate is on the template and punch the wells with the help of gel puncher. 5. Add 10 ml of the standard antigen and test antigen samples are to the wells. 6. Pour 1X TBE buffer into the electrophoresis tank such that it just covers the gel. 7. Run the Electrophoresis at 80-120 volts and 60-70 mA, until the antigen travels 3-4 cms from the well. 8. Incubate the glass plate in a moist chamber overnight at 37o C and results interpreted. 9. In case positive for reaction, the tips of the precipitin peaks are marked and the peak height measured from the upper edge of the well to the tip of the peak. 10. A graph is plotted of the rocket height (on Y-axis) versus the concentration of antigen (on X-axis) on a semi-log graph sheet. The concentration of the unknown is determined from the graph by finding the concentration against the rocket height. RESULT A precipitation ‘rocket’ spreading out from the loading well indicate positive reaction or specific antigen-antibody reaction due to the presence of antibody specific to the antigen. The absence of the precipitation indicates no reaction or the absence of any corresponding antibody – antigen. The height of the rocket, and its area are directly proportional to the amount of antigen in the sample, that is, the height of the precipitin peak depends on the concentration of antigens loaded in the corresponding wells.
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