Several enzyme-activity based methods are commercially available for the measurement of Thymidine Kinase 1 (TK1). Here we compare them with the measurement of TK1 protein concentration using the AroCell TK 210 ELISA. We will demonstrate how the application of ELISA technologies has increased the value and applicability of TK1 as a biomarker for the study of cancer and its therapies.
The traditional method for measuring TK1 is to measure its enzyme activity. This can be done in several different ways by using different substrates. In the ”gold-standard” assay TK1 activity is estimated by measuring the phosphorylation of the DNA nucleoside, deoxythymidine to dTMP (deoxythymidine monophosphate) with ATP as a substrate (Sharif et al., 2012). This assay has been commercialized later as TK-REA by using 125I-deoxyuridine instead of deoxythymidine.
Several published studies have shown the clinical value of TK-REA for the prognosis and monitoring therapy of different malignancies. There are several disadvantages of the traditional radioassay and TK-REA including that the tests are radioisotope-based, relatively expensive, time consuming, require specialized training and facilities, are relatively inconsistent, and have relatively low sensitivity (F. Zhang et al., 2001).
Another commercial assay for measuring the TK enzyme activity is TK-Liaison which utilizes azidothymidine (AZT) as a substrate and measures the AZT-phosphate product using a competitive ELISA. The assay exploits the advantages of sensitivity and accuracy associated with an ELISA compared with a radioisotope end point (Öhrvik et al., 2004). A third method for measuring TK1 activity is the DiviTum assay that uses bromo-deoxyuridine (BrdU) as a substrate (Nisman et al., 2013). DiviTum and TK-Liaison assays are very similar although the DiviTum is a manual assay and the Liaison is automated. These assays correlate at higher TK1 activities but not at low ones. It has been suggested that this difference may be due to the DiviTum assay being less sensitive to the TK1 forms found in the serum of normal subjects (Nisman et al., 2013). Both the assays showed potential in predicting post-operative recurrence in breast cancer patients (Nisman et al., 2013).
A general demerit of TK1 enzyme activity assays is that serum may contain TK1 inhibitors and that TK1 activity assays are less sensitive for the low molecular weight complexes found in the serum of subjects with solid tumors (Jagarlamudi et al., 2015). This may be why their application has been mainly restricted to the study of hematological malignancies.
AroCell started development of a commercial ELISA for determining TK1 protein concentrations in 2009 and the AroCell TK 210 ELISA was launched in 2012 for Research Use Only. The assay has been further developed and obtained CE accreditation in September 2015. Prior to the AroCell’s development of the AroCell TK 210 ELISA, other attempts were made to develop serum TK1 ELISAs, but none are CE accredited (Elfagieh et al., 2012; Alegre et al., 2014).
Studies have shown that the AroCell TK 210 ELISA correlates well with the gold-standard TK1 assay in hematological malignancies but has a higher sensitivity in sera from solid tumors (Kiran Kumar et al., 2016). In a recent study, AroCell TK 210 ELISA was compared with TK-Liaison and it was found that AroCell TK 210 ELISA had a similar sensitivity to TK-Liaison in sera from patients with hematological malignancies. However, in case of solid tumors, the AroCell TK 210 ELISA had higher sensitivity compared to TK-Liaison (0.37 vs 0.26) at the specificity of 0.96 (Un published data).
This increased sensitivity is due to the lack of interference found in ELISA assays and the use of a specific monoclonal antibody (TK 210) with the ability to detect the enzymatically inactive TK1 forms which are found in sera from subjects with solid tumors. Furthermore, a unique pre-treatment buffer releases TK1 from its serum complexes and makes it more available for assay.
Another advantage compare to activity based assays, is the absence of cross-reaction with TK2 and non-human TK1 forms making the assay more sensitive and specific for in vitro and animal studies. This increases the value of TK1 as a translational biomarker.
The replacement of complex enzyme-activity TK1 assays with their lack of specificity and sensitivity with a standard, robust and sensitive protein concentration ELISA method has made it a more valuable and more widely applicable biomarker in cancer disease management and drug discovery.
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