Abstract. Continuously monitoring cell cultures is essential for both controlling critical parameters and improving understanding of key processes. An ideal technique in this context is surface plasmon resonance (SPR) spectroscopy, which essentially exploits changes in the angle of incident light that occur when molecules bind to a surface. It provides the ability to monitor real-time changes in small concentrations of various molecules, with no need for additional labels or sample preparation. Here we present an SPR-based immunoassay for monitoring concentrations of human serum albumin (HSA), and compare its sensitivity when used in conjunction with a Biacore platform and the cheaper, smaller li SPR system. In conjunction with either system, the immunoassay can detect HSA (a hepatocyte viability marker) at concentrations typically present in three-dimensional hepatocyte cultures mimicking the liver used to evaluate effects of drug candidates before exposure to humans or animals. Furthermore, in conjunction with the li SPR system, it is sufficiently sensitive to measure the much lower HSA levels present in skin-hepatocyte co-cultures.

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

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.

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