Cell-Based ELISA Protocol
ELISA, or enzyme-linked immunosorbent assay, is used as a diagnostic tool in medicine and in biological research to quantify the amount of a specific protein. ELISA uses antibodies to detect the protein of interest and a reagent that produces a color. The amount of color relates to the amount of protein.
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Cell Culture
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Cells should be placed into the wells of a 96-well plate with 100 micro-liters of growth media and the cells left to settle overnight. If you're studying the cells' reaction to a factor, cells should be serum-starved for 24 hours and then stimulated with the factor under study.
Fixation and Primary Antibody
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After removal of the media, the cells are treated with a fixative such as 3 percent formaldehyde, or methanol. The cell membrane needs to be permeabilized to allow the antibody to penetrate the cell. Permeabilization can be achieved by the use of 0.1 percent Triton, if methanol has not previously been used. Nonspecific binding can be prevented with the use of 10 percent fetal calf serum diluted in phosphate-buffered saline. After removal of the block, the primary antibody can be applied and incubated for one hour.
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Secondary Antibody and Stain
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Remove the primary antibody, and wash three times. Add a secondary antibody linked to a detection agent, such as horseradish peroxidase. Incubate for an hour. Wash. Add chemiluminescent substrate solution. Develop and scan the plate for color intensity.
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References
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