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What are the steps involved in a colorimetric direct ELISA?

Posted July 21, 2023


The steps involved in a colorimetric direct ELISA (enzyme-linked immunosorbent assay) are as follows:

  • Coat ELISA plate with the antigen of interest, seal plate and incubate the plate overnight at 4°C
  • Remove coating solution and wash plate 2 times with desired buffer
  • Block plate with desired blocking solution for a specific time (e.g., 1-2 hours) at 4°C to block any remaining uncoated sites
  • Wash the plate several times with a wash buffer (e.g., PBS with a detergent like Tween-20) to remove unbound primary antibody and other nonspecific components.
  • Prepare a suitable substrate solution specific to the enzyme conjugated to the primary antibody (e.g., chromogenic substrate for horseradish peroxidase, HRP).
  • Incubate the plate at room temperature for 1-2 hours to allow the enzyme-substrate reaction to occur.
  • Add a stop solution (e.g., sulfuric acid for TMB substrate) to halt the enzymatic reaction and stabilize the color development.
  • Measure the absorbance signal at 650 nm with an ELISA microplate reader

These steps outline the basic procedure for a colorimetric direct ELISA. It's important to note that the exact conditions, reagents, and incubation times may vary depending on the specific assa. Always refer to the detailed protocols or instructions provided by the assay manufacturer or established protocols in scientific literature for specific guidance.

Additional resources

Enzyme-Linked Immunosorbent Assay (ELISA)

Screen Quest™ Colorimetric ELISA cAMP Assay Kit

Antibodies 101: ELISA (Enzyme-linked Immunosorbent Assay)