Table 1 Summary of TK1 assays and reported results
From: Thymidine kinase 1 through the ages: a comprehensive review
TK1 assay | Brief description | Measurement units | Normal versus cancer |
|---|---|---|---|
[3H] Deoxythymidine phosphorylation | Samples are incubated with ATP, DTT, MgCl2 and [3H]-labeled deoxythymidine. Labeled thymidine converted by TK1 to [3H]-dTMP is quantified to determine TK1 levels based on standard curves | pmol/min/mL | Normal: 0.6–3.1 pmol/min/mL Cancer: 0.9–48 pmol/min/mL [127] |
TK-radio enzymatic assay (TK-REA) | Assay utilizes [125I]-IUdR, an analog substrate of TK1, to measure TK1 activity. The output of this assay gives linear turnover of substrate for cellular dTK-F (TK1). 1 unit enzyme = 1.2 × 10–18 kat | U/L | *Normal: 2.5 × 10–1 U/L *Low-grade malignancy: 4.6 U/L *Intermediate grade: 28.8 U/L *High grade: 45 U/L [88] |
TK1-Liaison | 3-Azido-3-deoxythymidine (AZT) is converted to AZTMP by TK1. Phosphorylation is quantified through a competitive ELISA using anti-AZTMP antibody and AZTMP-labeled peroxidase. Color development is inversely proportional to TK1 activity and reported as U/L | U/L | Normal: 2.4–5.6 U/L Cancer: 4.0–8.2 U/L [114] |
DiviTum™ | BrdU is converted to BrdUMP by TK1 and then to BrdUTP by yeast enzymes. The dUTP formed during the reaction is washed over immobilized polyA strands. BrdUTP is detected with an anti-BrdU-antibody conjugated to alkaline phosphatase. The color development is measured at 405 nm | Divitum units (Du)/L | Normal: 9–33 Du/L Cancer: 20–92 Du/L [114] |
Chemiluminescent dot blot (IgY) | A nitrocellulose membrane is blotted with ~ 3 µL of sample and dilutions of a TK1 standard peptide. The membrane is probed with anti-TK1 chicken antibody (IgY). Blot intensities are measured using a charge-coupled device camera. A standard curve is used to infer sample measurements | pM | Normal: < 2.0 pM [124] |
TK1-210 | Antibodies used in this sandwich ELISA are designed against the exposed C-terminal end of TK1. Monoclonal antibodies against TK1 are immobilized on a microtiter plate and used to capture TK1 peptides. Secondary antibody conjugated to biotin is used to detect TK1. Strept-HRP is used to develop color (absorbance 450 nm) | ng/mL | Normal: 0.17–0.33 ng/mL Cancer: 0.17–9.9 ng/mL [127] |