A New Sandwich ELISA for Quantification of Thymidine Kinase 1 Protein Levels in Sera from Dogs with Different Malignancies Can Aid in Disease Management

Abstract

Thymidine kinase 1 (TK1) is a DNA precursor enzyme whose expression is closely correlated with cell proliferation and cell turnover. Sensitive serum TK1 activity assays have been used for monitoring and prognosis of hematological malignancies in both humans and dogs. Here we describe the development of a specific sandwich TK1-ELISA for the quantification of TK1 protein levels in sera from dogs with different malignancies. A combination of rabbit polyclonal anti-dog TK1 antibody and a mouse monoclonal anti-human TK1 antibody was used. Different concentrations of recombinant canine TK1 was used as standard. Clinical evaluation of the ELISA was done by using sera from 42 healthy dogs, 43 dogs with hematological tumors and 55 with solid tumors. An established [3H]-dThd phosphorylation assay was used to determine the TK1 activity levels in the same sera. The mean TK1 activities in dogs with hematological tumors were significantly higher than those found in healthy dogs. In agreement with earlier studies, no significant difference was observed in serum TK1 activities between healthy dogs and dogs with solid tumors. However, the mean TK1 protein levels determined by new TK1-ELISA were significantly higher not only in hematological tumors but also in solid tumors compared to healthy dogs (mean ± SD = 1.30 ± 1.16, 0.67 ± 0.55 and 0.27± 0.10 ng/mL, respectively). Moreover, TK1-ELISA had significantly higher ability to distinguish lymphoma cases from healthy based on receiver operating characteristic analyses (area under the curve, AUC, of 0.96) to that of the activity assay (AUC, 0.84). Furthermore, fluctuations in TK1 protein levels during the course of chemotherapy in dogs with lymphoma closely associated with clinical outcome. Overall, the TK1-ELISA showed significant linear correlation with the TK1 activity assay (r s = 0.6, p<0.0001). Thus, the new TK1-ELISA has sufficient sensitivity and specificity for routine clinical use in veterinary oncology.

Evaluation of serum thymidine kinase 1 activity as a biomarker for treatment effectiveness and prediction of relapse in dogs with non‐Hodgkin lymphoma

“…These results are in concordance with previous studies suggesting that in treated dogs with increased sTK1 activity at diagnosis, sTK1 activity drops to minimal level when the clinical remission has been achieved. 49,[53][54][55]73 One limitation in this study is that dogs were not completely restaged at the time of their best clinical response and sTK1 activity measurement to confirm the CR status. In 9 of 36 (25%) dogs classified as being in complete remission, the sTK1 activity remained above 20 Du/L at the time of their best response.…”

The Current State of Clinical Application of Serum Biomarkers for Canine Lymphoma

“…A subsequent report confirmed that the early results could be recapitulated in a different population using a non-radiometric immunoassay with high correlation ( 16 ). This assay has shown promise as a biomarker of disease, prognosis, and monitoring ( 13 , 17 – 19 ). The commercial application of this assay will be discussed subsequently.…”

Novel monoclonal antibodies against thymidine kinase 1 and their potential use for the immunotargeting of lung, breast and colon cancer cells

“…It is also known that TK1 can form different complexes. Some of these complexes in serum having abnormal molecular weights of up to 240 kDa and 730 kDa [30][31][32][33]. Therefore, antibodies targeting only one region of the TK1 protein may limit the detection of TK1 to specific forms of the enzyme.…”