How to Perform an ELISA Assay

An ELISA, or enzyme-linked immunoabsorbent assay, is a biochemical lab technique done to detect an antigen (such as a protein) or antibody in an experimental sample. The assay takes advantage of antibody-antigen binding and can be used to assay other cell and molecular binding mechanisms as well.

In a protein-based ELISA assay, a "capture" antibody or protein is laid down in an assay dish and allowed to adhere to the dish, after which the "probe" agent (such as one's protein of interest) is laid down and incubated with the capture agent. After several washes, a "detection" antibody is added in order to visualize the presence or absence of antibody-antigen or protein-protein binding.

Things You'll Need

• Assay dish (for example, 96-well block)
• Capture antibody (diluted to 5 ug/mL in 50 mM NaHCO2, pH 9.6)
• Probe antibody/protein (diluted to various concentrations)
• Detection antibody (for example, horseradish peroxidase-coupled antibody)
• PT buffer (1x PBS, 0.05 percent Tween-20)
• Blocking buffer (1x PBS with 5 mg/mL BSA, or 5 percent skim milk in 1x PBS)
• Detection reagent (ie. TMB reagent mixture)
• Mechanical lab shaker
• Cold room or refrigerator
• Pipetter and tips

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Instructions

• 1

  Dilute capture antibody to 5 ug/mL in 50 mM NaHCO2 (pH 9.6). Add 50 uL to each well of the sample dish used for testing. Cover with plastic or aluminum and incubate overnight at 4 degrees C on a shaker.

• 2

  Dump out capture antibody and wash two times with 200 uL PT buffer. Add 200 uL of blocking buffer per well (PB or 5 percent skim milk). Incubate anywhere from two hours to overnight while shaking at 4 degrees C.

• 3

  Dump out blocking buffer and wash 4 times with 200 uL PT buffer. Add "probe" protein samples at various concentrations in PB or 5 percent skim milk, adding 100 uL per well. Incubate one hour shaking at 4 degrees C. Dump out probe solutions and wash six times with 200 uL PT buffer.

• 4

  Add detection antibody (HRP-conjugated antibody) diluted 1:4000 in PB buffer or 5 percent skim milk (either one containing 0.05 percent Tween-20), adding 50 uL per sample well. Incubate for 30 minutes to one hour shaking at 4 degrees C.

• 5

  Dump out detection antibody solution and wash 6 times with 200 uL PT buffer per well, followed by washing two times with 200 uL of 1x PBS (phosphate-buffered saline). Dump out PBS and add 100 uL of detection reagent per well(for HRP, use freshly-mixed TMB reagent by mixing TMB substrate and peroxide at 1:1).
  Incubate for 15 minutes at room temperature, shaking occasionally.

  When using TMB and HRP: Finally, add 100 uL of 1M H3PO4 (phosphoric acid) per well in order to quench the TBM reaction.

  Read plate using sample analysis reader, such as a 96-well plate reader. (For HRP and TMB, use the "ELISA End-Point Assay" template available on most readers.)