ELISA testing


Identifying an unknown viral pathogen requires using a transmission electron microscope because they are so small, much smaller than bacteria. But, if a virus is known and can be isolated, an immunological reaction can be induced in an animal such as mice or rabbits. Using, then, antibodies from such a reaction can be used to detect the presence of the virus or viroid in infected tissue or liquid. This is the basic principle of the Enzyme-Linked-ImmunoSorbant-Assay, commonly referred to as the ELISA test, a serological test. ELISA is used in medicine to detect viruses such as HIV in people and is used in agriculture such as to detect potato viruses such as PVX, PVY and PLRV. It is the 'state of the art' for seed certification. State seed Certification Agencies and Associations as the one in Nebraska use ELISA to detect viruses in seed tuber lots in the 'winter test' conducted in Florida and other warmer climates. ELISA is quick and can detect viruses even in the absence of any plant symptoms of disease.

Some viruses cause diseases with clear symptoms such as leaf roll and calico, and may be readily identified in the field. However, an important virus as PLRV may infect a plants late in the season via transmission by green peach aphids and, although may not show symptoms, may be enough as to cause a severe problem in the next generation. Therefor, extracting sap from tubers harvested from seed and using ELISA will detect any latent viruses. Identifying PVX, PVY, etc. can not easily be done in the field and ELISA is needed.

How does ELISA work? ELISA plates (having 96 wells) are available for each specific virus. Each well contains the virus-specific anti-body bound to its sides. Sap or liquid extracted from tissue or cells is added to the well. In order to minimize possible over-reactions or unwanted reactions, and to titer the virus, the liquid may be diluted several times with a buffer. If the virus is present in the test liquid, it will bind to its anti-body. Wells are rinsed to remove the liquid and its contents that did not bind and therefor not the targeted virus. More of the same anti-body bound to the well is added. This second set of anti-bodies also has an enzyme attached to it which will react with a pigment. These anti-bodies attach to the viruses held by the bound anti-body. After this second reaction, any unattached anti-body is rinsed away. And now, the pigment substrate is added. If the substrate attaches to the enzyme because it is present, it will develop or change color. A color change means the targeted virus is present in the sap or tissue extract and if no change occurs than the virus is absent.

Quick Summary:

wells with bound anti-body ->
add test liquid & rinse: virus binds to anti-body ->
add anti-body having enzyme & rinse: if virus present, anti-body attaches ->
add enzyme substrate-dye: if virus present, dye attaches to enzyme and reacts.