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General procedure and tips for ELISA assay requiring a secondary conjugated antibody.
For accurate quantitative results, always compare signal of unknown samples against those of a standard curve. Standards (duplicates or triplicates) and blank must be run with each plate to ensure accuracy.
Coating antigen to microplate
Incubation with primary and secondary antibody
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 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. Be aware that the substrate is light sensitive so keep and store it in the dark.
ABTS (2,2'-azino-di-[3-ethyl]-bensothiazoline-6 sulfonic acid) diammonium salt.
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 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.
View more of our ELISA resources.