The Enzyme-Linked Immunosorbent Assay (ELISA) is a universally used analytical biochemical assay to detect the presence of antibody otherwise antigen from a sample. It has been used as a diagnostic tool in medical and plant pathology, as well as a quality-control check in various industries.
ELISA is a plate-based assay technique designed for detecting and quantifying antigen as well as antibody. In this process, a specific antigen or antibody must be immobilized to a solid surface and then complexed with an antibody or an antigen that is linked to an enzyme. If anyone performs an ELISA to detect antigen, then it is called sandwich ELISA. On the other hand, antibody can be detected by indirect ELISA. In Advanced Seed Research & Biotech Centre (ASRBC), ACI Ltd. laboratory, both type of ELISA is used to detect and quantify pathogen as well as antibody. There are some common viral diseases in potato like, Potato Leaf Roll Virus (PLRV), Potato Mop Top Virus (PMTV), Potato Virus S (PVS), Potato Virus X, Potato Virus Y (PVY) etc. which reduce plant growth, yield, and economic value of potato as well as it can impact the production of certified seed. So, to produce virus-free plantlet, it is vital to ensure the working materials are virus-free. That is why in ASRBC plant pathology laboratory, sandwich ELISA is used to detect the potato viruses. On the other hand, in ACI Animal Health Diagnostic Laboratory, both indirect and sandwich ELISA are in practice to antibody (like IBD, IBV, Fowl Adenovirus Antibody etc.) and antigen (like Avian influenza, Reovirus infections, hepatitis etc.) detection respectively.
The basic principle of this technique is antigen-antibody binding, including an enzyme-labeled antigen or antibody and enzyme activity is measured colorimetrically.
It begins with a coating step, in which the first layer, consisting of a target antigen or antibody, is adsorbed onto a 96-well polystyrene plate. This is followed by a blocking step in which all unbound sites are coated with a blocking agent. Following a series of washes, the plate is incubated with enzyme-conjugated antibody or antigen. Another series of washes remove all unbound antibody or antigen. A substrate is then added that measure the enzyme activity by producing a calorimetric signal only in presence of antibody or antigen (what for ELISA has been performed) in samples. Light absorption of the product formed after substrate addition is measured and converted to numeric values.
Prepared by
Aqief Afzal
Research Assistant
ASRBC, ACI Limited
