ELISA (enzyme linked immunosorbent assay)

ELISA is a tried and true technology borrowed from clinical immunology where a combination of antibodies, chemicals, and samples can yield results visible by eye. There are a range of different ELISA formats and chemistries available which are used by Agdia Testing Services. The most common format used is what we call a DAS ELISA (double-antibody sandwich enzyme linked immunosorbent assay).

To perform this test, we add your sample to a specially prepared test well on a microtiter plate. The inside of the test well is coated with an antibody that captures and holds particles of the virus we're testing for.  After the sample has been incubated for a period of time, it will be washed out of the test well.  Any analyte captured by the antibodies coated to the well, will remain intact.

We then add an enzyme conjugate, which is a two-part substance made up of an antibody chemically joined to an enzyme. If any virus particles are present, held in place by the first antibody, some of the enzyme conjugate sticks to them and remains in the well when we wash it out.

Then we add a colorless substrate that the enzyme conjugate can break down into a colored one. If enzyme conjugate is present, attached to virus particles, color appears in the test wells.  Positive samples will vary in intensity depending on how much analyte or virus particles were present and captured. Negative wells will not react with the substrate, leaving the wells virtually clear.

After this substrate incubation the plate is read by a spectrophotometer and by eye. The spectrophotometer provides a numberic value that correlates with the amount of color in the well.  Higher values indicate more color and a higher analyte concentration.  Negative samples generate very low values that fall into a statistically different numeric range than positive samples.

Every time we perform a DAS ELISA test, we also test a positive control, a sample from a plant we know to be infected. Checking that the positive control well is colored helps us know the test worked properly.