A Fluorescent Glycolipid-Binding Peptide Probe Traces Cholesterol Dependent Microdomain-Derived Trafficking Pathways

10.1371/journal.pone.0002933PLoS ONE38

Surface plasmon resonance based detection of human serum albumin as a marker for hepatocytes activity

Techniques for monitoring cell cultures and fermentation processes not only enable prompt feedback to variations in critical parameters (e.g., media composition and metabolites) but further improve our understanding of the processes themselves. In this context, surface plasmon resonance (SPR) spectroscopy is one of the methods of choice. This technique exploits angle shifting to follow molecular interactions in real-time. Therefore, it allows samples to be characterized without additional molecular labels and time-consuming sample preparation. The immobilization of receptors onto the chip surface is one of the most challenging requirements in SPR. Especially for measurements in crude samples, it is crucial to achieve a sufficient immobilization level and block the remaining sensitive area to prevent nonspecific binding. In this article, we present a SPR-based detection system for human serum albumin (HSA). As HSA is exclusively synthesized in the liver, it can be used to characterize the specific activity of in vitro cultivated human hepatocytes. These can be cultivated in so-called multi-organ-chips, which have been developed by groups at the TU Berlin and Fraunhofer IWS for predictive preclinical substance evaluation

Kinetic Analyses of Data from a Human Serum Albumin Assay Using the liSPR System

We used the interaction between human serum albumin (HSA) and a high-affinity antibody to evaluate binding affinity measurements by the bench-top liSPR system (capitalis technology GmbH). HSA was immobilized directly onto a carboxylated sensor layer, and the mechanism of interaction between the antibody and HSA was investigated. The bivalence and heterogeneity of the antibody caused a complex binding mechanism. Three different interaction models (1:1 binding, heterogeneous analyte, bivalent analyte) were compared, and the bivalent analyte model best fit the curves obtained from the assay. This model describes the interaction of a bivalent analyte with one or two ligands (A + L ↔ LA + L ↔ LLA). The apparent binding affinity for this model measured 37 pM for the first reaction step, and 20 pM for the second step