• Melissa Fernard Bøg Pedersen
Approximately 2-5% of all pregnancies are affected by preeclampsia, the cause of which remains unknown. Patients with established preeclampsia have decreased Plasminogen Activator Inhibitor-2 (PAI-2) plasma levels, indicating that PAI-2 might have a role in the development of preeclampsia, or that the decrease in PAI-2 levels might be a result of preeclampsia. PAI-2 has the ability to spontaneously form polymers, which could be relevant in a preeclampsia perspective. Thus, the possible role of PAI-2 in the etiology of preeclampsia needs to be investigated in full.
Therefore, the aim of this study was to develop experimental methods, models and tools to aid in the investigation of PAI-2 and its role in preeclampsia. These methods aimed to evolve on existing methods to induce polymerisation of PAI-2 and models to visualise the degree of polymer formation in samples. In addition, the aim herein was to generate and characterise Monoclonal Antibodies (mAbs), with the intent to establish an ELISA with the purpose of quantifying of the levels of PAI-2 in plasma samples from pregnant individuals.
In vitro models for polymerisation of PAI-2 were established. The polymerisation was induced chemically and thermally, using exposure to Guanidine Hydrochloride (GuHCl) or heat, respectively. These samples were then visualised using SDS-PAGE, native PAGE and western blotting techniques. Mouse mAbs towards PAI-2 were characterised and a new generation of rat mAbs against PAI-2 was generated and partially characterised. An ELISA based assay was established, with a mouse mAb as the capture antibody, and a pAb to detect captured PAI-2 from samples. This assay was used on plasma samples from preeclamptic patients and controls. Moreover, PAI-2 in pregnancy plasma samples was investigated, utilising magnetic DynaBeadsTM with a mouse mAb, to capture PAI-2 from the sample. The results of this were illustrated with western blotting techniques. Lastly, anti-PAI-2 mAbs were raised in rats, in an attempt to create high-affinity antibodies with different specificities, which might be applicable in further optimisation of ELISA and for the detection of PAI-2 polymers.
The results from in vitro polymerisation of PAI-2, induced chemically and and heat, showed polymerisation in the samples. Furthermore, PAI-2 in plasma samples showed intact, non-polymerised PAI-2 and polymerised PAI-2. The established ELISA for quantification of PAI-2 levels in plasma samples from preeclampsia patients compared with controls, showed detectable amounts of PAI-2. The rat mAbs were able to detect PAI-2, when used for western blotting applications. Furthermore, they could bind both intact and polymerised PAI-2, when PAI-2 samples were bound on an ELISA plate. However, none of these antibodies seemed to have specificity towards only polymerised PAI-2. The ELISA based assay established in this study remains to be validated. Additionally, rat mAbs must be characterised in their ability to recognise native, human PAI-2 from plasma samples. Future studies must be designed to investigate if there is a difference in the amount of PAI-2 polymers present in plasma from patients affected by preeclampsia.
LanguageEnglish
Publication date2021
External collaboratorUniversity of Southern Denmark
Yaseelan Paralasah ypalarasah@health.sdu.dk
Other
ID: 433972980