Development of affinity chromatography for calcium-binding proteins

Pre-eclampsia (PE) is a common and severe pregnancy complication and is associated with substantial perinatal morbidity and mortality in mothers and infants. The disease is often characterized by a non-specific presentation which makes it challenging for physician to diagnose PE during regular pregnancy check-ups. To date, there are no diagnostic tests on the market for detection of PE early in pregnancy (first trimester). In this project, we will develop a platform to sensitively analyse calcium-binding proteins (CBPs) which will unlock the full potential of CBPs as predictive PE markers. The technology will also be applicable for other diseases (e.g., dementia and cancer) where CBPs are also known to play a key role in disease pathophysiology. We will develop with phage display antibodies that can recognize calcium binding to specific motifs in proteins. To this end we will synthesize peptide motifs with and without calcium to select antibodies that are specific for calcium bound proteins. These antibodies will be validated for their clinical use. For this goal we will use serum samples from the Improved studie (EU subsidised study) to determine if we can recognize pre-eclampsia in a very early stage. This knowledge can lead to a better treatment of pregnant women suffering from this disease and also will probably increase the well-being for the baby born and the development further in life.

We have made in collaboration within the Erasmus MC two phage display libraries a human scFv phage library and a nanobody phage library (cancer cell line injected in a llama). We tried to acquire selected antibodies and nanobodies by this phage display procedure. We encountered the problem that if specific peptides of a protein were used covalently linked to a carrier protein (ovalbumin) antibody binders were obtained. However the antibodies were especially raised to the linker used to covalently bind the peptide to the carrier protein. We were not able to select in these libraries antibodies to specific calcium bound proteins by this bait procedure with ovalbumin. Probably larger fragments or whole proteins are necessary to reach the goal to select nanobodies or antibodies or to use baits without the linker structure what is used to bind a specific peptide to a carrier protein. We have searched contact with a German Chemical Company to help us in this respect.