Fundamental Principle of ELISA

Enzyme linked immunosorbent essay (ELISA) is a biochemical technique used mainly in immunology to detect the presence of antibody or antigen in a sample. The Elisa has been used as diagnostic tool in medicine and plant pathology as well as quality control check in various industries.

Principle: Unknown amount of antigen (protein) is affixed to the surface and then specific antibody is washed over the surface, so that it can bind to specific antigen. The antibody-antigen complex is flooded with enzyme that can convert the complex to some detectable signal. For example, in case of fluorescence ELISA, when light is shown upon the sample, antigen/antibody complexes will fluoresce so that amount of antigen in the sample can be measured.

Direct ELISA: antibody is the protein that immune system makes. Elisa plate is 96 well plate that has special surface which binds protein. When put protein sample in the plate, the protein will stick to the wall. When detection antibody with enzyme for color change is added, these antibody will stick to specific protein of interest, showing color change. The extra unbound antibody is washed away.Indirect ELISA: indirect ELISA makes use of 2 antibody instead of one. Primary detection antibody sticks directly to the protein of interest , but there is no enzyme stuck to it. Then after, secondary antibody with enzyme to produce color change is added, that will stick to first detection antibody.Sandwich ELISA: In sandwich ELISA, we add capture antibody before we put sample (protein) so that onl antibodies will stick to the plate. Excess antibodies is washed away and empty surface is blocked by using blocking protein (BSA or detergent). Now our sample is introduced into the plate, where only the protein of interest only will be sticked while other antigen sill not be sticked to the antibody. This is more sensitive in the sense that all of the protein of interest will be captured and others will be washed away. Now detection antibody with enzyme for color change is introduced for detection of reaction.