No abstract

Substrates for plasmonic sensors in a flow-through configuration are mostly fabricated by cost-intensive clean room processes, whereas high-volume diagnostic devices are typically made of polymers. This contrast could limit the application of this efficient flow regime in mass-produced devices. In order to become more compatible with polymer processing, a commercially available polycarbonate filter membrane has been evaluated as a substrate for plasmonic flow-through biosensing. The membrane has been sputtered with gold and its sensitivity to changes of bulk refractive index has been determined by transmission measurements using sodium chloride solutions. The sensitivity has been evaluated by determining the wavelength barycenter in a wavelength interval from 470 to 800 nm. The highest determined sensitivity to variations in bulk refractive index is 117 nm RIU −1 (refractive index units). This sensitivity is smaller than that of regular arrays of nanoholes. But the integrating character of the applied evaluation leads to an average standard deviation of 0.005 nm which results in a resolution of 4.1 • 10 −5 RIU. This resolution is sufficient for the detection of protein adsorptions. The proof of principle has been shown with bovine serum albumin and a simplified immunoassay, which consists of the sequential addition of protein A, an IgG antibody and its corresponding antigen. The results show the applicability of this polymeric membrane for biosensing applications. These substrates could enable plasmonic sensing in a flow-through configuration in disposable diagnostic devices.

Monoclonal antibodies are produced in cultured hybridoma cell lines, but these cells tend to be unstable; it is therefore necessary to rescue the corresponding genetic information. Here we describe an improved method for the amplification of antibody variable gene (V-gene) information from murine hybridoma cells using a panel of specific, non-degenerate primers. This primer set allows sequences to be rescued from all murine V-genes, except the lambda light chain genes, which rarely contribute to murine immune diversity. We tested the primers against a range of antibodies and recovered specific amplification products in all cases. The heavy and light chain variable regions were subsequently joined by a two-step cloning strategy or by splice overlap extension PCR.

SNAP-tag technology allows recombinant proteins to be covalently labeled to O(6)-benzylguanine (BG)-modified substrates with 1:1 stoichiometry. By attaching according fluorophores, this method is ideally suited for in vitro and in vivo imaging, as well as protein interaction analyses. Fluorophores modified with BG react with the SNAP-tag, whereas those modified with O(2)-benzylcytosine (BC) conjugate to a more recent derivative known as the CLIP-tag. The orthogonal substrate specificity of the SNAP- and CLIP-tags extends the range of applications by allowing double labeling. We previously developed a monoclonal antibody (mAb) that recognizes both tags. In this study, we describe a new mAb, which is specific for the SNAP-tag alone. Therefore, this mAb allows discrimination between SNAP- and CLIP-tags within a broad range of immunological methods, including enzyme-linked immunosorbent assays, western blotting, flow cytometry, and immunohistochemistry.