Q.2. (c) Discuss ELISA assay for antibody by indirect methods and antigen by sandwich method. Enzyme Linked ImmunosorbentAssay (ELISA)
Ans.2. (c)The steps of the general, "indirect," ELISA for determining serum antibodyconcentrations are:
1. Apply a sample of known antigen of known concentration to a surface, often thewell of a microliter plate. The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. These samples of known antigen concentrations will constitute a standard curve used to calculate antigen concentrations of unknown samples. Note that the antigen itself may be an antibody.
2. A concentrated solution of non-interacting protein, such as bovine serum albumin (BSA) or casein, is added to all plate wells. This step is known as blocking, because the serum proteins block nonspecific adsorption of other proteins to the plate.
3. The plate wells or other surface are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. Since antigen immobilization in this step is due to nonspecific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards.
4. The plate is washed, and a detection antibody specific to the antigen of interest isapplied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.
5. Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substratespecificenzyme. This step may be skipped if the detection antibody is conjugated to an enzyme.
6. Wash the plate, so that excess unbound enzyme antibody conjugates are removed.
7. Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.
8. View/quantify the result using a spectrophotometer, spectrofluorometer, or other optical/electrochemical device.
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Sandwich ELISA for antigen:A less common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:
1. Prepare a surface to which a known quantity of capture antibody is bound.
2. Block any nonspecific binding sites on the surface.
3. Apply the antigen containing sample to the plate.
4. Wash the plate, so that unbound antigen is removed.
5. Apply primary antibodies that bind specifically to the antigen.
6. Apply enzyme linked secondary antibodies which are specific to the primary antibodies.
7. Wash the plate, so that the unbound antibodyenzymeconjugates are removed.
8. Apply a chemical which is converted by the enzyme into a colour or fluorescent or signal.
9. Measure the absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.
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