ELISA was developed in 1971 by two groups independently, namely Engvall and Perlmann, and Werman and Schuurs. Later Vollar and Clark and Adams utilized ELISA for detection of virus infection.
Principle: Also known as E.I.A, it is the most sensitive technique used for detecting the antigen or antibody. The principle used here is "The ability of low molecular weight antibody to couple with enzyme to produce enzymatically active immunological conjugates and the immune reaction can be detected through histochemical staining techniques." It is an enzyme-substrate reaction and when exposed to colored substrate complexes, releases color. Such a substrate is a chromogenic substrate. The intensity of the color is measured by taking O.D using a colorimeter.
Procedure: Three types of ELISA techniques are wildly used namely Sandwich ELISA, Indirect ELISA and Competitive ELISA.
1) A monoclonal antibody preparation that will recognize the antigen to be diagnosed is attached to the surface of a microtitre well.
2) The test sample is coated onto this.
3) If the antigen present in the test sample is homologous to the antibody; binding occurs to form antigen-antibody complex.
4) If the antigen is not present, no binding occurs.
5) After a wash, all unbound materials are removed.
6) A second preparation of the monoclonal antibody is prepared such that it has the enzyme attached to it. This is added to the microtitre well.
7) The substrate is then added and the colored reaction product is measured.
1) Coating of the antigen onto the surface of a microtitre well.
2) The test antiserum is added and allowed to incubate.
3) If the antibody is homologous, it will bind to the antigen. Enzyme is added to the antibody-antigen complex. The substrate is added and the colored reaction product is measured.
1) Antibody is first incubated in solution with a sample containing antigen.
2) Microtitre well is coated with antigen.
3) Antigen- antibody mixture is added. Enzyme is added. After the addition of substrate, color is measured.
Applications: Sandwich method is used to test HEP-B.
This is mainly used for separating protein portions from serum samples. Immunoelectrophoresis mainly consists of two methods namely immunodiffusion and electrophoresis.
Electrophoresis is defined as the migration of charged particles under the influence of an electric field which facilitates the separation of a mixture of a compound into its component parts. In immunodiffusion the separated components (antigens) obtained by electrophoresis are allowed to diffuse through agar.
Procedure: The agar plate loaded with human serum (antigen) is placed on the support of electrophoresis apparatus. Two filter paper strips at two ends of the agar plate. The other ends of the filter paper strip are dipped into the filter paper strip are dipped into the buffer contained in the trough of electriphoretic apparatus. An electric current of 3 to 6 volt/cm is applied. Electrophoretic separation is achieved in 45 minutes. After 45 minutes the current is stopped and agar plate is taken out. After electrophoretic separation of human serum the antiserum is loaded into the antiserum trough by using a micropipette. After that the agar plate is placed in a moisture chamber. This allows immunodiffusion to take place. The electrophoretically separated proteins (antigen) and antibody diffuse towards one another. When they meet antibody-antigen complexes are formed. They are marked by precipitin curves at the points of confluence.
1) To test proteins present in urine and body fluids.
2) To test normal and abnormal serum proteins.
3) For identification of serum proteins.
Counter current Immunoelectrophoresis
This is also an immunological technique which is much related to immunoelectrophoresis. Here the antigen and antibody are kept in separate wells in an agar plate and they are allowed to migrate simultaneously on agar plate by applying an electric field. The gammaglobulin in gel with alkaline provides the antibody a positive charge and it migrates towards the cathode. The antigen attains a negative charge and it migrates towards anode. They converge each other to form precipitin line in the middle.
In this type of immunoelectrophoresis, the antiserum is incorporated in the agar plate. The precipitin curve formed here has the appearance of a rocket and hence the name. At one end of the agar plate, antigens of increasing concentration are kept. On electrophoresis the antigen move towards the anode forming a rocket shaped precipitin are. The height of the rocket is propotional to the antigen concentration. By comparing the standard rocket height with that obtained from the experiment, the amount of antigen present in serum can be obtained.
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