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Immunoelectrophoresis
Immunoelectrophoresis is a technique that combines electrophoresis and immunodiffusion to separate and characterize proteins based on their charge and reaction with antibodies. It involves electrophoresing an antigen mixture to separate components by charge, cutting troughs in the gel for antiserum, and detecting lines of precipitation where antibodies and antigens meet. Immunoelectrophoresis is used qualitatively in clinical laboratories to detect the presence or absence of proteins in serum and identify normal and abnormal proteins. It can detect immunodeficiencies or overproduction of proteins but is limited for quantitative analysis.
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Immunoelectrophoresis
- 1. Dr. J. P.Saranraj IMMUNOELECTROPHORESIS
- 2. IMMUNOELECTROPHORESIS Immunoelectrophoresis wasfirst coined by Grabar and Williams in 1953. Immunoelectrophoresis is a general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies. Immunoelectrophoresis is the combination of Immunodiffusion (Mancini’s Single Radial Immunodiffusion and Ouchterlony Double Diffusion) and Electrophoresis.
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- 6. IMMUNOELECTROPHORESIS In Immunoelectrophoresis,the antigen mixture is first electrophoresed to separate its components by charge. Troughs are then cut into the agar gel parallel to the direction of the electric field, and antiserum is added to the troughs. Antibody and antigen then diffuse toward each other and produce lines of precipitation where they meet in appropriate proportions. Immunoelectrophoresis is a strictly Qualitative technique that only detects relatively high antibody concentrations (greater than 100 g/ml), it utility is limited to the detection of quantitative abnormalities only when the departure from normal is striking, as in immunodeficiency states.
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- 8. Applications of Immunoelectrophoresis Immunoelectrophoresis is used in clinical laboratories to detect the presence or absence of proteins in the serum. Detection of Immunodeficiency (In Immunodeficiency sample, no precipitin band is formed with particular antigen). Detection of Over production of Serum proteins (Albumin, Immunoglobulin andTransferrin). Detection of deficiency in Complement. Used to identify normal and abnormal proteins in Urine or Serum. Testing the purity ofAntigen.
- 9. ROCKET ELECTROPHORESIS Arelated Quantitative technique, Rocket electrophoresis, does permit measurement of antigen levels. Also called as Laurell Technique or One dimension electroimmunodiffusion. It is called as an adaption of SRID and more rapid than SRID. In Rocket electrophoresis, a negatively charged antigen is electrophoresed in a gel containing antibody. The precipitate formed between antigen and antibody has the shape of a Rocket (measured), the height of which is proportional to the concentration of antigen in the well.
- 10. One limitationof rocket electrophoresis is the need for the antigen to be negatively charged for electrophoretic movement within the agar matrix. Some proteins, immunoglobulins for example, are not sufficiently charged to be quantitatively analyzed by Rocket electrophoresis.