elisa test

elisa test





Testing plant samples for virus using the ELISA technique - photo tour

The first step is to add an antibody to a specific virus to the ELISA plates. Commercial ELISA test kits with antibodies for all major soybean viruses are available. We use antibodies for four common soybean viruses: alfalfa mosaic, soybean mosaic, bean leaf mottle and tobacco streak virus.

Next we prepare sap from plant samples to be tested for virus. We grind the plant tissue with a phosphate buffer. This is the same grinding procedure we use in the plant indicator test for viruses.The sap is loaded into the coated plates. If a specific virus is present, it will bind to the antibodies.

A positive control and a negative control is included in each ELISA plate.
The positive control is sap obtained from plants known to be infected with the target virus.
Sap from a healthy plant is used as a negative control.

A map is used to keep track of the location of each sample as we fill the plate. This is a good place to jot down important notes about the sample.

The plates are incubated overnight, and then emptied. A quick flip of the wrist empties the wells without contamination.

Another chemical is added that will react with the enzyme attached to antibody. The resulting color will give an indication if virus was present in the plant sap. In this case, the yellow wells indicate samples collected from plants infected with Bean pod mottle virus (BPMV).

An automatic plate reader is used to quantify ELISA results, based on the color reaction.

Values that are the sum of the mean plus 4 times the standard deviation of the negative values are considered positive (generally above 0.1)
Reference: Crowther, J.R. 1995. Methods in Molecular Biology: ELISA Theory and Practice. Totowa, NJ: Humana Press.