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 substrate and 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 ) .
