TITLE: Sandwich ELISA Protocol
AUTHORS: Sam Li

[DESCRIPTION]
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[STEPS]
?. [Coat the Plate:]
Dilute unlabeled capture antibody to a final concentration of 0.5-8µg/ml in Coating Buffer (BioLegend, Cat. No. 421701) and transfer 100µl to each well of a high affinity, protein-binding ELISA plate (e.g., BioLegend Cat. No. 423501).
?. [Coat the Plate:]
Seal plate to prevent evaporation. Incubate at 4°C overnight.
?. [Block the Plate:]
Bring the plate to room temperature, flick off the capture antibody solution, wash 3 times with PBS/Tween, and block non-specific binding sites by adding 200µl of Blocking Solution to each well.Note: You may need to experiment with different blocking solutions, such as gelatin or milk, to find one that will give you the lowest background noise.
?. [Block the Plate:]
Seal plate and incubate at room temperature for 1 hour.
?. [Block the Plate:]
Wash 3 times with PBS/Tween. Firmly blot plate against clean paper towels.
?. [Add Standards and Samples:]
Dilute standards and samples to desired concentrations in Blocking Solution (perform dilutions in polypropylene tubes or plate) and add 100µl per well to the ELISA plate.Note: Try to match the diluent of the standards as closely as you can to the matrix in your samples. For example, if your samples are cell culture supernatants, use the same media to dilute the standards.
?. [Add Standards and Samples:]
Seal the plate and incubate at room temperature for 2-4 hours or at 4°C overnight.
?. [Add Standards and Samples:]
Wash 3 times with PBS/Tween. Washes can be effectively accomplished by filling wells with either a squirt bottle, carboy, manifold dispenser, multi-channel pipettor or automatic plate washer. For increased stringency, after each wash, let the plate stand briefly, flick off the buffer, and blot plates on  paper towels before refilling.Note: It is very important to use clean paper towels between each wash. This will help avoid any possible cross-well contamination.
?. [Add Detection Antibody:]
Dilute the biotin-labeled detection antibody to 0.25-2µg/ml in Blocking Solution. Add 100µl of diluted antibody to each well.
?. [Add Detection Antibody:]
Seal the plate and incubate at room temperature for 1 hour.
?. [Add Detection Antibody:]
Wash 3 times with PBS/Tween.
?. [Add Detection Antibody:]
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 100µl per well.
?. [Add Detection Antibody:]
Seal the plate and incubate at room temperature for 30 minutes.
?. [Add Detection Antibody:]
Wash 5 times with PBS/Tween.
?. [Add Substrate (ABTS for slower color development):]
Thaw ABTS Substrate Solution within 20 min of use. Add 11µl of 30% H2O2 per 11 ml of substrate and vortex. Immediately dispense 100µl into each well and incubate at room temperature (4-60 min) for color development. To stop the color reaction, add 50µl of ABTS Stop Solution.
?. [Add Substrate (ABTS for slower color development):]
Read the optical density (OD) for each well with a microplate reader set to 405 nm.
?. [OR Add Substrate (TMB for faster color development):]
For each plate, mix 6 ml of TMB Reagent A with 6 ml TMB Reagent B (BioLegend TMB Substrate Reagent Set, Cat. No. 421101) immediately prior to use. Transfer 100µl into each well. Incubate at room temperature (4 – 30 min) for color development. To stop the color reaction, add 100µl of TMB Stop Solution.
?. [OR Add Substrate (TMB for faster color development):]
Read the optical density (OD) for each well with a microplate reader set to 450 nm.