Sandwich ELISA protocol

General procedure and tips for sandwich ELISA.

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A sandwich ELISA measures antigen between two layers of antibodies (capture and detection antibody). The target antigen must contain at least two antigenic sites capable of binding to antibodies.

Monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in sandwich ELISA systems. Monoclonal antibodies recognize a single epitope that allows quantification of small differences in antigen. A polyclonal is often used as the capture antibody to pull down as much of the antigen as possible.

Sandwich ELISAs remove the sample purification step before analysis and enhance sensitivity (2–5 times more sensitive than direct or indirect).

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General note

Sandwich ELISA procedures can be difficult to optimize and tested match-paired antibodies should be used. This ensures the antibodies are detecting different epitopes on the target protein and do not interfere with the other antibody binding. We are unable to guarantee our antibodies in sandwich ELISA unless they have been specifically tested.

Review antibody datasheets for tested applications information.

Coating with capture antibody

  1. Coat the wells of a PVC microtiter plate with the capture antibody at 1–10 μg/mL concentration in carbonate/bicarbonate buffer (pH 9.6).

    Unpurified antibodies (eg ascites fluid or antiserum) may require increased concentration of the sample protein (try 10 μg/mL) to compensate for the lower concentration of specific antibody.
  2. Cover the plate with adhesive plastic and incubate overnight at 4°C.
  3. Remove the coating solution and wash the plate twice by filling the wells with 200  μL PBS. The solutions or washes are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel.

Blocking and adding samples

  1. Block the remaining protein-binding sites in the coated wells by adding 200 μL blocking buffer (5% non-fat dry milk/PBS) per well.
  2. Cover the plate with adhesive plastic and incubate for at least 1–2 h at room temperature or overnight at 4°C.
  3. Wash the plate twice with 200 µL PBS.
  4. Add 100 μL of diluted samples to each well. Always compare signal of unknown samples against those of a standard curve. Run standards (duplicates or triplicates) and blank with each plate. Incubate for 90 min at 37°C.

    Ensure concentration of standards spans the most dynamic detection range of antibody binding. You may need to optimize the concentration range to obtain a suitable standard curve. Always run samples and standards in duplicate or triplicate.
  5. Remove samples and wash the plate twice with 200 μL PBS.

Incubation with detection and secondary antibody

  1. Add 100 μL of diluted detection antibody to each well.

    Check that the detection antibody recognizes a different epitope on the target protein to the capture antibody. This prevents interference with antibody binding. Use a tested matched pair whenever possible.
  2. Cover the plate with adhesive plastic and incubate for 2 h at room temperature.
  3. Wash the plate four times with PBS.
  4. Add 100 μL of conjugated secondary antibody, diluted in blocking buffer immediately before use.
  5. Cover the plate with adhesive plastic and incubate for 1–2 h at room temperature.
  6. Wash the plate four times with PBS.


Horse radish peroxidase (HRP) and alkaline phosphatase (ALP) are the two most widely used enzymes for detection in ELISA assays.

Consider that some biological materials have high levels of endogenous enzyme activity (such as high ALP in alveolar cells, high peroxidase in red blood cells) that may result in nonspecific signal. If necessary, perform an additional blocking treatment with levamisol (for ALP) or 0.3% H2O2 in methanol (for peroxidase).

ALP substrate
P-Nitrophenyl-phosphate (pNPP) is the most widely used substrate for most applications. Measure the yellow color of nitrophenol at 405 nm after 15–30 min incubation at room temperature and stop the reaction by adding equal volume of 0.75 M NaOH.

HRP chromogenes
The substrate for HRP is hydrogen peroxide. Cleavage of hydrogen peroxide is coupled to oxidation of a hydrogen donor which changes color during reaction.

TMB (3,3’,5,5’-tetramethylbenzidine)
Add TMB solution to each well, incubate for 15–30 min, add equal volume of stopping solution (2 M H2SO4) and read the optical density at 450 nm.

OPD (o-phenylenediamine dihydrochloride)
The end product is measured at 492 nm. Keep and store the substrate it in the dark as it is light sensitive.

ABTS (2,2’-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt)
The end product is green and the optical density can be measured at 416 nm.

Always handle with care and wear gloves as some enzyme substrates are considered hazardous (potential carcinogens).

Data analysis

Prepare a standard curve from the serial dilutions data with concentration on the x axis (log scale) vs absorbance on the Y axis (linear). Interpolate the concentration of the sample from this standard curve.

Other useful resources

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