Detection
Although many different types of enzymes have been used for detection, horse radish peroxidase (HRP)
and alkaline phosphatase (ALP) are the two most 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 a nonspecific
signal. If necessary, perform an additional blocking treatment with Levamisol (for ALP) or with 0.3%
solution of H2O2 in methanol (for peroxidase).
ALP substrate
For most applications pNPP (p-Nitrophenyl-phosphate) is the most widely used substrate.The yellow colour
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).
HRP chromogenes
The substrate for HRP is hydrogen peroxide. Cleavage of hydrogen peroxide is coupled to oxidation of a
hydrogen donor which changes colour 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. Be aware that the
substrate is light sensitive so keep and store it in the dark.
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.
Note: some enzyme substrates are considered hazardous (potential carcinogens), therefore always handle
with care and wear gloves.
Step 14.
Dispense 100 μl (or 50 μl) of the substrate solution per well with a multichannel pipet or a multipipet.