IMMUNOELECTROPHORESIS

IMMUNOELECTROPHORESIS

• 1.
  IMMUNOELECTROPHORESIS
• 2.
   Immunoelectrophoresis refersto precipitation in agar under an electric field.  It is a process of combination of immuno-diffusion and electrophoresis.  An antigen mixture is first separated into its component parts by electrophoresis and then tested by double immuno-diffusion.  Antigens are placed into wells cut in a gel (without antibody) and electrophoresed. A trough is then cut in the gel into which antibodies are placed.  The antibodies diffuse laterally to meet diffusing antigen, and lattice formation and precipitation occur permitting determination of the nature of the antigens.  The term “Immunoelectrophoresis” was first coined by Grabar and Williams in 1953.
• 3.
   When electriccurrent is applied to a slide layered with gel, antigen mixture placed in wells is separated into individual antigen components according to their charge and size.  Following electrophoresis, the separated antigens are reacted with specific antisera placed in troughs parallel to the electrophoretic migration and diffusion is allowed to occur.  Antiserum present in the trough moves toward the antigen components resulting in formation of separate precipitin lines in 18-24 hrs, each indicating reaction between individual proteins with its antibody.  Immunoelectrophoresis is used in both clinical and research laboratories for separating and identifying proteins on the basis of their electrophoretic behaviour and their immunological properties.  Proteins such as rabbit serum proteins, which are antigens, when injected into another animal such as a goat (the host), elicit the production of antibodies in the host.  The interaction between the antigen and its antibody, which is also a protein, is both strong and highly specific. If solutions of antigen and antibody are mixed in different ratios, it is found that at a specific ratio, known as the equivalence point, the binding is maximized and a precipitate of antigen-antibody complex is formed.  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.
• 5.
  Agarose gel isprepared on a glass slide put in a horizontal position. Using sample template, wells are borne on the application zone carefully. The sample is diluted 2:3 with protein diluent solution (20μl antigen solution +10 μl diluent). Using a 5 μl pipette, 5 μl of control and sample is applied across each corresponding slit (Control slit and Sample slit). The gel is placed into the electrophoresis chamber with the samples on the cathodic side, and electrophoresis run for 20 mins/ 100 volts. After electrophoresis completes, 20 μl of the corresponding antiserum is added to troughs in moist chamber and incubated for 18- 20 hours at room temperature on a horizontal position. The agarose gel is placed on a horizontal position, and dried with blotter sheets. The gel in saline solution is soaked for 10 minutes and the drying and washing repeated twice again. The gel is dried at a temperature less than 70°C and may be stained with protein staining solution for about 3 minutes followed by decolorizing the gel for 5 minutes in distaining solution baths. The gel is dried and results evaluated.
• 6.
  RESULT • Presence ofelliptical precipitin arcs represents antigen-antibody interaction. • Absence of formation of precipitate suggests no reaction. • Different antigens (proteins) can be identified based on the intensity, shape, and position of the precipitation lines.