In the first step, TK1 present in the patient sample phosphorylates AZT to the corresponding 5'-monophosphate (AZTMP).
AZT and its monophosphate derivative, AZTMP, are ideal candidates against which antibodies could be raised.
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. Alternatively, the procedure may be modified by replacing the assay buffer with a sample diluent.
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.