Sandwich ELISA Protocol
Dilute unlabeled capture antibody to a final concentration of 0.5 – 8 µg/ml in Coating Buffer (BioLegend, Cat. No. 421701) and transfer 100 µl to each well of a high affinity, protein-bindingELISA plate (e.g., BioLegend Cat. No. 423501).
Seal plate to prevent evaporation.
Incubate at 4°C overnight.
Bring the plate to room temperature, flick off the capture antibody solution.
Seal plate and incubate at room temperature for ≥ 1 hour.
Firmly blot plate against clean paper towels.
Dilute standards and samples to desired concentrations in Blocking Solution (perform dilutions in polypropylene tubes or plate) and add 100 µl per well to the ELISA plate.
Seal the plate and incubate at room temperature for 2-4 hours or at 4°C overnight.
Wash with PBS/Tween (1/3).
Wash with PBS/Tween (2/3).
Wash with PBS/Tween (3/3).
Dilute the biotin-labeled detection antibody to 0.25 – 2 µg/ml in Blocking Solution.
Add 100 µl of diluted antibody to each well.
Seal the plate and incubate at room temperature for 1 hour.
Wash with PBS/Tween (1/3).
Wash with PBS/Tween (2/3).
Wash with PBS/Tween (3/3).
Dilute the Av-HRP conjugate (Cat. No. 405103) or other enzyme conjugate to its pre-determined optimal concentration in Blocking Buffer (usually between 1/500 – 1/2000).
Add 100 µl per well.
Seal the plate and incubate at room temperature for 30 minutes.
Wash with PBS/Tween (1/5).
Wash with PBS/Tween (2/5).
Wash with PBS/Tween (3/5).
Wash with PBS/Tween (4/5).
Wash with PBS/Tween (5/5).
Thaw ABTS Substrate Solution within 20 min of use.
Add 11 µl of 30% H2O2 per 11 ml of substrateand vortex.
Immediately dispense 100 µl into each well and incubate at room temperature (4-60minutes) for color development.
To stop the color reaction, add 50 µl of ABTS Stop Solution.
Read the optical density (OD) for each well with a microplate reader set to 405 nm.
Wash with PBS/Tween (1/3).
Wash with PBS/Tween (2/3).
Wash with PBS/Tween (3/3).
Block non-specific binding sites by adding 200 µl of Blocking Solution to each well.
Wash with PBS/Tween (1/3).
Wash with PBS/Tween (2/3).
Wash with PBS/Tween (3/3).