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Premium member Presentation Transcript IMMUNOELECTROPHORESIS: IMMUNOELECTROPHORESIS INSTRUMENTAL METHODS OF ANALYSIS Vasanthan V -IEP-Synopsis: Electrophoresis Immunoelectrophoresis Immunodiffusion Principle of I mmunoelectrophoresis Types Instrumentation Conclusion Applications SynopsisElectrophoresis : Electrophoresis , also called cataphoresis , is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field . During electrophoresis, molecules placed in an electric field acquire a charge and move towards appropriate electrode . The mobility of molecule is dependent on a numbers of factors: 1.Size/shape of molecule 2.pH 3.Heat generated by ionic strength buffer 4.Charge particles ElectrophoresisImmunoelectrophoresis : Immunoelectrophoresis is a general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies . All variants of I mmunoelectrophoresis require immunoglobulins , also known as antibodies reacting with the proteins to be separated or characterized. Immunoelectrophoresis (IEP) combines two techniques, electrophoresis and immunodiffusion . ImmunoelectrophoresisImmunodiffusion : Immunodiffusion is a qualitative or semi-quantitative test based on the principles of double diffusion described by Oudin and Ouchterlony . An antibody and its homologous soluble antigen are placed in separate wells cut in a suitable diffusion medium ( agarose or CleargelTM ) and allowed to diffuse outward into the medium . Between the two wells, a concentration gradient of each of the reaction components is established ranging from antigen excess closest to the antigen well, to antibody excess closest to the antibody well. A visible line of precipitate forms at the point of equivalence. ImmunodiffusionSlide 6: ImmunodiffusionPrinciple of Immunoelectrophoresis : The principle of I mmunoelectrophoresis is the formation of precipitate lines at the equivalence point of the antigen and its corresponding antibody . Principle of I mmunoelectrophoresisTypes : Crossed I mmunoelectrophoresis Rocket I mmunoelectrophoresis Fused Rocket I mmunoelectrophoresis Affinity I mmunoelectrophoresis TypesInstrumentation : In this method it is important that the ratio between the quantities of antigen and antibody be correct (antibody titer ). When the antibody is in excess, statistically at most one antigen binds to each antibody while when the antigen is in excess at most one antibody binds to each antigen. Yet at a specific antigen/antibody ratio (equivalence point) huge macromolecules are formed. InstrumentationSlide 10: They consist of an antigen-antibody-antigen-antibody-... s equence and are immobilized in the gel matrix as an I mmuno precipitate . The white precipitate lines are visible in the gel and can be revealed with protein stains . The method is specific and the sensitivity is very high because distinct zones are formed.Slide 11: In Two-dimensional immunoelectrophoresis . Antigens are separated on the basis of electrophoretic mobility. The second separation is run at right angles to the first which drives the antigens into the antiserum-containing gel to form precipitin peaks; the area under the peak is related to the concentration of antigen. Ab - Antibodies Ag- AntigensImmunoelectrophoresis -Antivenom: Immunoelectrophoresis - Antivenom Each antibody molecule can bind two separate sites on an antigen molecule (venom toxin), consequently antibodies have the ability to cross link many antigen molecules simultaneously. This cross-linking causes the antibody-antigen-complex to become insoluble and precipitate out from the solution. The I mmunoelectrophoresis technique makes use of this capability of the antibodies to form giant insoluble complexes with their respective antigens. The antigen-antibody precipitate which forms can be visualized by specific staining techniques, or quantified by various means.Immunoelectrophoresis can be divided into three principles: Counter Immunoelectrophoresis Zone E lectrophoresis/ Immunodiffusion The “Rocket ” technique Immunoelectrophoresis can be divided into three principlesA. Counter Immunoelectrophoresis: According to Bussard ( 1959) i n an agarose gel exhibiting high electro endosmosis , the buffer is set at a pH about 8.6 so that the antibody does not carry any net charge. The sample and the antibody are placed in their respective wells and move towards each other: the charged antigens migrate electrophoretically and the antibodies are carried by the electro-osmotic flow . A. Counter I mmunoelectrophoresisB. Zone Electrophoresis/Immunodiffusion: According to Grabar and Williams ( 1953) first a zone electrophoresis is run in an agarose gel , followed by the diffusion of the antigen fraction towards the antibody which is pipetted into troughs cut in the side parallel to the electrophoretic run. B. Zone Electrophoresis/ I mmunodiffusionC. The “Rocket” technique: According to Laurell (1966) and the related methods antigens migrate in an agarose gel which contains a definite concentration of antibody. As in method A(Counter) the antibodies are not charged because of the choice of the buffer . As the sample migrates one antibody will bind to one antigen until the ratio of concentrations corresponds to the equivalence point of the immunocomplex . C. The “Rocket ” techniqueConclusion: Immunoelectrophoresis identifies proteins based on their combined E lectrophoretic and Immunological properties . This method is useful to monitor antigen and antigen-antibody purity and to identify a single antigen in a mixture of antigens. The three major I mmunoglobulins were detecting are Immunoglobulin M( IgM ), Immunoglobulin G( IgG ), and Immunoglobulin A (IgA ). C onclusionSlide 18: S erum proteins are separated by agarose gel Electrophoresis and the point of equivalence is observed by the maximum antigen-antibody complex formation . The properties of agarose gel shave a small number of fixed charges, which cause a phenomenon known as E lectroendosmosis (EEO ).Slide 19: EEO causes a slow net flow of water through the gel away from the positive electrode. At a pH of 8.5, antibodies are nearly uncharged , and their slow electrophoretic migration is nullified by the EEO flow through the agarose . Most other proteins are highly charged.Applications: IEP is used for the diagnosis and differential diagnosis of monoclonal gammopathies when using serum and urine specimens. The most common application of IEP is the diagnosis of monoclonal gammopathies . A monoclonal gammopathy is a condition in which a single clone of plasma cells produces elevated levels of a single class and type of immunoglobulin. The elevated Immunoglobulin is referred to as a monoclonal protein, M-protein, or paraprotein . Monoclonal gammopathies may indicate a malignancy such as multiple myeloma or macroglobulinemia . ApplicationsSlide 21: The method is also used for a number of other purposes, including screening for circulating immune complexes, characterization of cryoglobulinemia and pyroglobulinemia , recognition and characterization of antibody syndromes, and recognition and characterization of the various forms of dysgammaglobulinemias . IEP is a reliable and accurate method for routine protein evaluations, detecting both structural abnormalities and concentration changes.References: http:// www.scribd.com/doc/7503174/IMMUNOELECTROPHORESIS TITAN GEL- Immunoelectrophoresis Procedure --Helena Laboratories Basic Immunologic Procedures, Terry Kotrla , MS, MT(ASCP)BB ReferencesQueries: Queries You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.