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).

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.

Buffers and reagents

Coating with capture antibody

Blocking and adding samples

Incubating with detection and secondary antibodies

Detection

Data analysis

See direct ELISA protocol buffers and reagents.

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 (e.g. 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 an adhesive plastic and incubate for 2 h at room temperature, or 4℃ overnight. The coating incubation time and temperature may require some optimization.

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.

1. Block the remaining protein-binding sites in the coated wells by adding 200 μL blocking buffer per well.

2. Cover the plate with adhesive plastic and incubate for at least 1–2 h at room temperature or overnight at 4℃. The blocking incubation time and temperature may require some optimization.

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℃. The ncubation time and temperature may require some optimization.

Ensure concentration of standards spans the most dynamic detection range of antibody binding.

5. Remove samples and wash the plate twice with 200 μL PBS.

1. Add 50-100 μL of detection antibody to each well, diluted at the optimal concentration (according to the manufacturer's instructions) in blocking buffer immediately before use...

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 an adhesive plastic and incubate for 2 h at room temperature. This incubation time and temperature may require optimization. Although 2 hours is usually enough to obtain a strong signal, if a weak signal is obtained, stronger staining will often observed when incubated overnight at 4℃.

3. Wash the plate four to five times with PBS.

4. Add 50-100 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 to five times with PBS.

1. Dispense 50-100 μL of the substrate solution per well with a multichannel pipet or a multipipet.

2. After sufficient color development (if it is necessary) add 100 μL of stopping solution to the wells.

3. Read the absorbance (optical density) of each well with a plate reader.

Note: some enzyme substrates are considered hazardous (potential carcinogens), therefore always handle with care and wear gloves. Although many different types of enzymes have been used for detection, horse radish peroxidase (HRP) and alkaline phosphatase (ALP) are the two widely used enzymes employed in ELISA assay. It is important to consider the fact that some biological materials have high levels of endogenous enzyme activity (such as high ALP in alveolar cells, high peroxidase in red blood cells) and this may result in non-specific signal. If necessary, perform an additional blocking treatment with levamisol (for ALP) or with 0.3% solution of H2O2 in methanol (for peroxidase).

For most applications pNPP (p-Nitrophenyl-phosphate) is the most widely used substrate. The yellow color of nitrophenol can be measured at 405 nm after 15-30 min incubation at room temperature. (This reaction can be stopped by adding equal volume of 0.75 M NaOH).

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

Add TMB solution to each well, incubate for 15-30 min, add equal volume of 2 M H2SO4 and read the optical density at 450 nm.

The end product is measured at 492 nm. Be aware that the substrate is light sensitive so keep and store it in the dark.

The end product is green and the optical density can be measured at 416 nm.Some enzyme substrates are considered hazardous (potential carcinogens), therefore always handle with care and wear gloves.

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.