5-11. CONJUGATE ENZYMES
Conjugates are enzyme-labeled antigen or antibody that retains activity capable
of converting the substrate into a product that can be easily detected. The enzymes
most frequently used for conjugates include horseradish peroxidase (HRP) and alkaline
a. The most commonly used conjugate enzyme in the EIA test procedures is
horseradish peroxidase which has the capability of reacting with a wider array of highly
chromogenic substances and is produced from a vegetable source.
b. The second most commonly used conjugate enzyme is alkaline phosphatase
which has a greater resistance to environmental toxins and is derived from the mucosa
of the calf intestines.
A substrate is a substance upon which an enzyme acts. 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) and para-nitrophenyl phosphate (p-NPP).
a. Ortho-phenylenediamine, the most satisfactory and commonly used
peroxidase substrate, yields a highly chromogenic orange product after enzyme
degradation has occurred.
b. Para-nitrophenyl phosphate is the substrate used for the enzyme alkaline
phosphatase and yields a highly chromogenic yellow-colored product after enzyme
degradation. Both of the above mentioned substrates are highly soluble and extremely
5-13. CLINICAL APPLICATIONS
Clinical application of EIA procedures include but are not limited to quantitation
and/or qualitation of serum components and various hormones. They also serve as
clinical tools in the diagnosis, evaluation, and treatment of the many autoimmune and