Generic (Quantitative) Sandwich ELISAΒΆ

Contributed by Martin Fitzpatrick, University of Birmingham, United Kingdom

A generic ELISA protocol with optional quantitative step.

  • Dilute primary antibody in coating buffer solution allowing for 50ul/well. A total of 5ml/96 well plate will provide spare for pipetting inaccuracy.
  • Coat Nunc Maxisorp plate with primary antibody solution at 50ul/well. Tap gently to ensure the bottom of each well is completely covered.
  • Incubate plate overnight at 4’C
  • Flick off primary antibody and blot plate on tissue.
  • Block plate with 200ul blocking buffer/well. A total of 20ml/96 well plate.
  • Incubate the plate at room temperature for 1hr
  • For quantitative ELISA prepare standards of target antigen in culture medium.

Perform serial dilutions from high of 2000pg/ml down to 31.25pg/ml, with final 1x blank culture medium alone.

  • Wash the plate x6 with PBS/1% Tween. After final wash tap out plate and blot dry.
  • Add supernatants (and optional standards) at 50ul/well. A 1/10 dilution plate can be produced by adding 45ul wash buffer + 5ul supernatant.
  • Incubate at room temperature for 2-4 hours
  • Flick off samples and standards into Vercon for disposal
  • Wash the plate x6 with PBS/1% Tween. After final wash tap out plate and blot dry.
  • Dilute secondary biotinylated antibody in blocking buffer to a volume for 50ul/well (5ml/96 well plate).
  • Add 50ul of diluted secondary antibody/well
  • Incubate at room temperature for 1-2 hours
  • Flick off secondary antibody to disposal
  • Wash the plate x6 with PBS/1% Tween. After final wash tap out plate and blot dry.
  • Dilute Extravidin peroxidase with blocking buffer at 1/1000 to a total volume of 50ul/well (5ml/96 well plate).
  • Add extravidin peroxidase to plate at 50ul/well
  • Incubate at room temperature for 1/2 hour
  • Wash the plate x8 with PBS/1% Tween. After final wash tap out plate and blot dry.
  • Add 100ul TMB substrate per well (10ml/96 well plate)
  • Leave at room temperature for ~20 minutes or until top standard has saturated.
  • Stop reaction with 1M HCl at 100ul/well
  • Measure abosorbance on plate reader.

TMB dual read 450nm with blank at 650nm