Sensitive Nonradiometric Method for Determining Thymidine Kinase 1 Activity

Abstract

AbstractBackground: Thymidine kinase 1 (TK1) is a cytoplasmic enzyme, produced only in the S-phase of proliferating cells, that has potential as a tumor marker. Specific determination of TK1 in serum is difficult, in part because of differences in the physical properties of serum TK1 compared with cytoplasmic TK1.Methods: The first step in the new assay was phosphorylation of 3′-azido-2′,3′-deoxythymidine (AZT) to AZT 5′-monophosphate (AZTMP) by TK1 present in patient material. The AZTMP formed was measured in a competitive immunoassay with specific anti-AZTMP antibodies and AZTMP-labeled peroxidase. Results were compared with those of a TK radioenzyme assay (REA) for 78 samples from patients suffering from hematologic diseases.Results: The detection limit was 78 μIU/L, and within-run CVs <20% were seen for samples with TK1 down to 130 μIU/L. Cross-determination of the mitochondrial isoenzyme TK2 activity was <0.1%. Between-assay imprecision (CV) was 3.5–7.4%, and the within-assay imprecision was 4.1–9.1%. In studies of recovery and linearity on dilution, measured values ranged from 84% to 115% of expected at concentrations of 0.26–10.4 mIU/L. Results of the new assay (mIU/L) = 0.109 × TK REA (U/L) + 0.092. Heterophilic antibodies did not interfere in the assay. The upper 95th percentile, in 100 healthy individuals, was 0.94 mIU/L, and the median value was 0.43 mIU/L.Conclusion: The TK1 enzyme-labeled immunoassay uses a stable substrate, is precise, appears to be accurate, and is resistant to interferences. It may provide a practical tool in the management of hematologic malignancies.