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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).
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.
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).
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.
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 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).
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.