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A sandwich ELISA measures the amount of antigen between two layers of antibodies (capture and detection antibody). The antigen to be measured must contain at least two antigenic sites capable of binding to antibody, since at least two antibodies act in the sandwich.
Either 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 fine detection and 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.
The advantage of sandwich ELISA is that the sample does not have to be purified before analysis, and the assay can be very sensitive (up to 2 to 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 so they do not interfere with the other antibody binding. Therefore, we are unable to guarantee our antibodies in sandwich ELISA unless they have been specifically tested for sandwich ELISA.
Please review antibody datasheets for information on tested applications.
Coating with capture antibody
The end product is green and the optical density can be measured at 416 nm.
Note: some enzyme substrates are considered hazardous (potential carcinogens), therefore always handle with care and wear gloves.
Dispense 100 μl (or 50 μl) of the substrate solution per well with a multichannel pipet or a multipipet.
Prepare a standard curve from the data produced from the serial dilutions 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.