The genetically and antigenically diverse group of noroviruses is the major cause of human viral epidemic gastroenteritis worldwide. Virus detection and control are thus crucial topics when aiming at containing and preventing the resulting large and often persisting outbreaks. Aptamers provide a promising alternative to antibodies concerning their ability to bind and thus detect and influence bio-active molecules. These small, single-stranded oligonucleotides are able to bind to a multitude of possible target molecules with high affinity. For a specific target the highest affinity aptamers are found by screening a randomized library. In this work a DNA aptamer capable of binding to the norovirus genotype II.4 capsid protein VP1 was found. The general approach is thereby not limited to norovirus capsid, but could be extended to almost any kind of biologically relevant molecule. The development of the library enrichment was further computationally analyzed in order to describe the enrichment during screening. This is the basis for a later extensive characterization of both target and aptamers that could lead to insights regarding the functional coherence of both partners. An abstract model describing this coherence could be utilized to generate a target-specific library, from which future aptamer screening runs could benefit.

Aptamers are synthetic single-stranded oligonucleotides which bind specifically to their target. They offer several advantages over antibodies. For example, aptamers can be produced under unphysiological conditions against almost any target, including toxic or pathological substances. They are also quicker and cheaper produced than antibodies, and are easy to modify without loss of activity. Furthermore, they exhibit high stability under a width range of conditions. Consequently, they make excellent receptors for the use in biosensors. This article describes the evaluation of a novel aptasensor based on the Surface Plasmon Resonance (SPR)-system developed by the Fraunhofer Institute for Applied Optics and Precision Engineering IOF (Jena, Germany) using a thrombin–aptamer interaction as a model system. The biotin-tagged aptamer was attached to the sensor's gold surface by means of its interaction with streptavidin. Thrombin solutions of different concentrations were pumped over this surface, and the interaction was measured under buffer flow. The binding signals for the thrombin–aptamer interaction were compared to those arising from a control random-oligonucleotide of the same size and bearing the same modifications. Using this approach, we were able to obtain reproducible, significant and stable signals with a limit of detection of about 26 nmol/L

Monoclonal antibodies have become an increasingly important part of fundamental research and medical applications. To meet the high market demand for monoclonal antibodies in the biopharmaceutical sector, industrial manufacturing needs to be achieved by large scale, highly productive and consistent production processes. These are subject to international guidelines and have to be monitored intensely due to high safety standards for medical applications. Surface plasmon resonance spectroscopy — a fast, real‐time, and label‐free bio‐sensing method — represents an interesting alternative to the quantification of monoclonal antibody concentrations by enzyme‐linked immunosorbent assay during monoclonal antibody production. For the application of monitoring bioactive and total monoclonal antibody concentrations in cell culture samples, a surface plasmon resonance assay using a target‐monoclonal antibody model system was developed. In order to ensure the subsequent detection of bioactive monoclonal antibody concentrations, suitable immobilization strategies of the target were identified. A significant decrease of the limit of detection was achieved by using an adapted affinity method compared to the commonly used amine coupling. Furthermore, the system showed limit of detection in the low ng/mL range similar to control quantifications by enzyme‐linked immunosorbent assay. Moreover, the comparison of total to bioactive monoclonal antibody concentrations allows analysis of antibody production efficiency. The development of an alternative quantification system to monitor monoclonal antibody production was accomplished using surface plasmon resonance with the advantage of low analyte volume, shorter assay time, and biosensor reusability by target‐layer regeneration. The established method provides the basis for the technical development of a surface plasmon resonance‐based system for continuous process monitoring.

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.

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.