In the second step, a competitive ELISA measures the amount of AZTMP by use of specific goat antibodies against AZTMP and HRP-labeled AZTMP.
Highly specific antibodies against AZT and AZTMP have been generated previously and used in clinical settings to determine the concentrations of these compounds in serum and extracts from tissues and cells (33, 39).
In human cells treated with AZT, activation by TK1 is a necessary step, and there is an accumulation of AZTMP because the next step catalyzed by thymidylate kinase (TMPK) is very inefficient (6,43).
Our polyclonal goat anti-AZTMP antibody was very specific for AZTMP, comparable to previously described rabbit antibodies (33).
Such cases can be tested by running the TK ELISA with or without filtration through a molecular weight cutoff filter before the detection of AZTMP.
Contrary to the TK REA assay, the ELISA assay can easily be standardized by calibration against AZTMP and use of a fixed conversion rate between IU/L and AZTMP formed in the assay, which is another major advantage of the new method.
A high AZT concentration probably makes the assay robust against interfering factors, and UMP was included to protect AZTMP from enzymatic degradation.