A monoclonal antibody-based sandwich ELISA for measuring canine Thymidine kinase 1 protein and its role as biomarker in canine lymphoma

• Introduction: Dogs play an important role in society, which increased during the covid epidemics. This has led to a much higher workload for the veterinarians. Therefore, there is a need for efficient diagnostic tools to identify risk of malignant diseases. Here the development of a new test that can solve some of these problems is presented. It is based on serum Thymidine Kinase 1 (TK1), which is a biomarker for cell proliferation and cell lysis.Methods: Anti-TK1 monoclonal antibodies were produced against two different epitopes, the active site of the TK1 protein and the C-terminal region of canine TK1. The antibodies were developed with hybridoma technology and validated using dot blot, Quartz Crystal Microbalance (QCM) technology, western blots, immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA). Clinical evaluation of Canine TK1 ELISA was done by using sera from 131 healthy dogs and 93 dogs with lymphoma. The two selected Anti-TK1 monoclonal antibodies have Kd values in the range of 10(-9) M and further analysis with dot and western blots confirmed the high affinity binding of these antibodies. A sandwich Canine TK1 ELISA was developed using the anti-TK1 antibodies, and TK1 concentrations in serum samples were determined using dog recombinant TK1 as a standard.Results: Serum TK1 protein levels were significantly higher in dogs with lymphoma compared to those in healthy dogs (p < 0.0001). Receiver operating curve analysis showed that the canine TK1-ELISA obtain a sensitivity of 0.80, at a specificity of 0.95. Moreover, the Canine TK1 ELISA has a positive predictive value (PPV) of 97%, and the negative predictive value (NPV) of 83%, reflecting the proportion of test results that are truly positive and negative. Furthermore, Canine TK1 ELISA had significantly higher capacity to differentiate dogs with T-cell lymphoma from those with B-cell lymphoma compared to earlier used TK1 activity assays.Discussion: These results demonstrate that the Canine TK1 ELISA can serve as an efficient tool in the diagnosis and management of dogs with lymphomas.

Ämnesord

LANTBRUKSVETENSKAPER  -- Veterinärmedicin -- Klinisk vetenskap (hsv//swe)

AGRICULTURAL SCIENCES  -- Veterinary Science -- Clinical Science (hsv//eng)

LANTBRUKSVETENSKAPER  -- Veterinärmedicin -- Annan veterinärmedicin (hsv//swe)

AGRICULTURAL SCIENCES  -- Veterinary Science -- Other Veterinary Science (hsv//eng)

LANTBRUKSVETENSKAPER  -- Veterinärmedicin -- Medicinsk biovetenskap (hsv//swe)

AGRICULTURAL SCIENCES  -- Veterinary Science -- Medical Bioscience (hsv//eng)

Nyckelord

canine TK1 ELISA

monoclonal antibody

immunoassay

serum TK1 activity

serum TK1 concentration

canine lymphoma

blood biomarker

tumor marker

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