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Assay development for cardiac troponin T and lactate dehydrogenase as markers for disease detection

Guo, Wei Li (2012) Assay development for cardiac troponin T and lactate dehydrogenase as markers for disease detection. PhD thesis, Dublin City University.

Human cardiovascular diseases (CVDs) are leading causes of human death globally and cause huge social and economic impacts. Similarly, bovine mastitis is the most economically important disease of dairy cattle world-wide. The pathogenesis of both diseases involves an inflammatory process. Biomarkers are measurable substances, which may be used to detect the presence of specific diseases. Cardiac troponin T (cTnT) is recommended as a potential biomarker for cardiovascular disease (CVD) detection due to its high specificity and sensitivity. In addition, elevation in L-lactate dehydrogenase (LDH) levels in mastitic milk suggests that it may have potential as a biomarker for the detection of sub-clinical mastitis. Classical CVD biomarkers, including cardiac myoglobin and creatine kinase-MB, have been extensively studied but are lacking in specificity and early–stage sensitivity. Troponin T was recommended as a potential ‘gold standard’ for CVD detection, and, therefore, a key aim of this project was to generate recombinant antibody fragments against cTnT and, ultimately, to develop a sensitive immunoassay for early-stage CVD diagnosis. Recombinant antibody fragments, including scFvs and Fabs, have excellent antigen recognition specificity but have additional benefits compared to standard antibodies. Current immunoassays for cTnT detection use monoclonal antibodies and the generation and application of recombinant antibodies and a novel assay platform may introduce significant improvements in cTnT assay development. A recombinant avian anti-cTnT scFv antibody was generated and the antigen binding capacity of the antibody was improved using chain-shuffling. The chain-shuffled antibody, with sub-nanomolar affinity, was subsequently incorporated into an ELISA inhibition assay for the detection of cTnT. Bovine mastitis is an inflammation of the mammary gland caused by invasion of pathogens. Its early detection is of paramount importance in controlling bovine health. Current diagnostic tests for mastitis have drawbacks such as high costs, lack of suitability for ‘on-site’ testing as well as poor specificity and sensitivity. Lactate dehydrogenase (LDH) was evaluated as a potential biomarker for the detection of sub- clinical mastitis in this research, LDH levels in milk samples were determined using a reliable and reproducible fluorescence-based assay, and the potential of LDH as a marker of subclinical mastitis was determined. It was found that LDH levels had a good correlation with somatic cell count, the gold standard mastitis marker. Moreover, it was shown that a number of factors including lactation stage and storage conditions affected LDH levels in milk samples. These factors must be taken into account when using LDH as biomarker for sub-clinical mastitis detection.
Item Type:Thesis (PhD)
Date of Award:November 2012
Supervisor(s):O'Kennedy, Richard
Uncontrolled Keywords:Human cardiovascular diseases; biomarkers; Recombinant antibody fragments
Subjects:Biological Sciences > Biotechnology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
ID Code:17175
Deposited On:20 Nov 2012 11:04 by Richard O'Kennedy . Last Modified 30 Jul 2021 14:48

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