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ELISA protocol
The University of Chicago





Basic ELISA Protocol

There are many different types of ELISAs, which can detect the presence of protein in serum or supernatent. One of the most common types of ELISA is the so-called "sandwich ELISA." It is termed this because the antibody that you are detecting gets sandwiched between an antigen and a chromogenically-conjugated antibody. Below you will find a basic protocol for this assay.

  • To coat the plate with the appropriate antigen, fill the microwells of a Nunc Maxi-Sorp Immuno Plate with 100uL of the diluted antigen.
  • Incubate at 4C overnight
  • Wash the unbound antigen off the plate by flicking the contents of the plate into the sink, fill the wells with DI water, flick again, repeat 2X with PBS-Triton.
  • Block non-specific binding by adding 200uL of 1%BSA/PBS
  • Incubate for 30-60minutes at Room Temperature (RT)
  • Wash plate as above
  • Add 100uL of (diluted) samples to appropriate wells. Be sure to include positive and negative controls, and, if necessary, a standard curve.
  • Incubate for 1hour at RT
  • Repeat washing step
  • Prepare appropriate dilution of the second step antibody conjugated either with Alkaline Phosphatase or Horseradish Peroxidase. (antibodies should be titrated for optimal dilution)
  • Add 100uL of second step antibody to wells and incubate for 1hr
  • Repeat washing step
  • Prepare substrate solution*
  • Add 100uL of substrate to well and incubate at RT for 60min
  • add stopping solution if appropriate
  • Read plates on an ELISA plate reader*

*Common Substrates and the appropriate plate reader setting

  • ABTS: 405-410 nm
  • TMB: non-stopped 620-650 nm, stopped 450 nm
  • OPD: non-stopped 450 nm, stopped 490 nm
  • pNPP: 405-410 nm
  • BluePhosTM: 595-650 nm