Elisa

aELISA (Enzyme-linked Immuno Sorbent Assay)

Which plant is transgenic?

DESCRIPTION:

ELISA is a method used to detect antigens. Its use is extended to all biological areas, including diagnostics, gene expression, antigenicity, etc.

It is based in the use of antibodies and its simplicity is admirable. We will be using ELISA to detect the presence of Neomycin Phosphotransferase (NPTII), an enzyme that provides antibiotic resistance to certain bacteria. This gene has been engineered as a selectable marker for transgenic plants. So when you produce transgenic plants with this marker, they will be resistant to Kanamycin.

The method will be able to detect the presence of NPTII in crude extracts of transgenic plants. ELISA uses one, two or three antibodies, one of them conjugated to an enzyme (usually horse radish peroxidase, HRP or alkaline phosphatase, AP) as a detection method. The choice of substrate will depend on your ability to detect the antigen in question. We will use a chromogenic substrate and we will compare to a standard curve of purified NPTII.

Pre-lab questions

1) What does ELISA means?

2) What is the main difference between monoclonal and polyclonal antibodies?

3) List three applications of the ELISA test

METHOD

Each group is going to work with a column (8 wells, from a 96 well plate format) and will prepare two to four extracts, one per plant (we may use more than two plants on a given experiment):

a) Plant A

b) Plant B

c) Plant C

PROTOCOL

1) Locate 3 microcentrifuge tubes label them A, B and C. Locate a column (with 8 wells) and bring it to your bench.

2) Label your column with the name of your team. Don't label the bottom, use the upper surface.

3) Produce 4 microcentrifuge punches or one or two leaves from each plant and place them in the microcentrifuge tube. Weight the tissue and write down the amount: Sample 1= __________mg, Sample 2= ___________ mg, etc.

4) Add 400 µl of the PEB1X...