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.

ELISA原理图 src="/upimg/allimg/070101/1251100.jpg" width=500>

• 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