Conjugates are enzyme-labeled antigen or antibody which retain activity capable
of converting the substrate into a product that can be easily detected.
The conjugates (enzymes) most frequently used include horseradish peroxidase
(HRP) and alkaline phosphatase (AP). The most commonly used conjugate in the
EIA test procedures is horseradish peroxidase.
The second most commonly used conjugate is alkaline phosphatase, which has a
greater resistance to environmental toxins and is derived from the mucosa of the
Substrates are chromogenic substances which are initially colorless but yield a
colored product after enzyme degradation.
The substrates most frequently used in the EIA procedures are:
Ortho-phenylenediamine (OPD), the most satisfactory and commonly used
peroxidase substrate, which yields a highly chromogenic orange product after
enzyme degradation has occurred.
Para-nitrophenyl phosphate (p-NPP), the substrate used for the enzyme alkaline
phosphatase and which yields a highly chromogenic yellow-colored product after
enzyme degradation. Both of the above mentioned substrates are highly soluble
and extremely light sensitive.
End of Lesson 5