(Enzyme-Linked ImmunoSorbant Assay)
ELISA is abbreviated term for Enzyme-Linked ImmunoSorbant Assay. This procedure is one of the most widely used methods in clinical immunology assays to detect the presence and absence of certain antigens or antibodies and also to quantify them when necessary. Quantification can be done in a range of microgram (µg) to nanogram (ng). The ELISA procedure takes advantage of the fact that most proteins will bind firmly to the surface of different kinds of plastic (polystyrene or polyvinyl chloride), usually by hydrophobic interaction.
The steps of the ELISA procedure are simple to perform. There are mainly FOUR phases in ELISA: 1.Coating & Blocking
There are mainly 3 methodologies in ELISA. The most frequently used methods are the indirect ELISA and sandwich ELISA because they are more sensitive in capturing of the measured compound (either antibody or antigen). Direct ELISA is less sensitive because there is higher chance of false results.
Indirect ELISA procedure:
1.Antigen bind to base (Coating phase)
2.Block unbound sites on plastic with unrelated proteins (Blocking phase) 3.Specific antibody (against that specific antigen) added. If quantification is needed, varying dilutions of the antibody are added in series. This antibody is called primary antibody. 4.Tertiary reagent added. This reagent usually consists of antibodies (labelled with enzyme) that will bind specifically to the bound antibodies in each well. This antibody is called secondary antibody. (Labelling phase)
The Bio-Rad Protein Assay, based on the method of Bradford, is a simple and accurate procedure for determining concentration of solubilised protein. It involves the addition of an acidic dye to protein solution, and subsequent measurement at 595 nm with a spectrophotometer or micro plate reader. Comparison to a standard curve provides a relative...