Abstract:
Disturbances of the natural balance between pro-coagulant and anticoagulant systems, due to hereditary or acquired factors may result in haemorrhagic or thrombotic diseases.
Currently the INR/ISI coagulation monitoring system, introduced in 1983 by the World Health Organisation (WHO), is that of choice for most anticoagulation management clinics. Patients undergoing anticoagulation therapy must regularly attend specialised outpatient clinics for the close monitoring and maintenance of their INR. The automated laboratory caters for rapid online simultaneous analysis of multiple blood samples, resulting in the calculation of a patient’s INR from the recorded prothrombin time. The insensitivity of the prothrombin time test has been well documented, requiring a reduction in the prothrombin concentration of 45 % prior to the materialisation of clinically significant prothrombin times.
The project aims to employ three avenues of biotechnology to aid in the development of an immuno or molecular imprinted polymer (MIP) based anticoagulation assay. The project will utilise computational molecular modelling in an attempt to visualise the tertiary structure of human prothrombin, which will allow the rational selection of antigenic sites for molecular imprinting or antibody production.
An aqueous, prothrombin-imprinted homo-polymer was grafted to the gold surface of a surface plasmon resonance biosensor (Biacore). The Biacore allowed the real time monitoring of imprinted polymer binding characteristics to i
homogeneous protein solutions. As a direct comparison of two technologies, molecular imprinting and immuno technology, polyclonal antibodies showing specificity towards the same prothrombin antigen were immobilised onto Biacore chips. The imprinted polymer graft and polyclonal antibody based assays recognised homogeneous solutions of prothrombin at a concentration range of 0.01 nM to 14.2 nM and 0.01 nM to 0.5 nM respectively. A randomised preliminary clinical trial was initiated to compare the two assays’ ability to differentiate plasma samples with a variety of INR values.
The results thus far show promise for the development of a new anticoagulation assay using molecularly imprinted polymer technology. The ultimate aim for this project is to develop a consistently more accurate point-of-care anticoagulation therapy monitoring kit, incorporating this new technology, which can replace or be used concomitantly with the INR/ISI system currently in use. This thesis raises more questions regarding the efficacy of oral anticoagulation therapy (OACT) and argues for and against the necessity of a novel OACT management assay.
