A lipid-binding loop of botulinum neurotoxin serotypes B, DC and G is an essential feature to confer their exquisite potency.

The exceptional toxicity of botulinum neurotoxins (BoNTs) is mediated by high avidity binding to complex polysialogangliosides and intraluminal segments of synaptic vesicle proteins embedded in the presynaptic membrane. One peculiarity is an exposed hydrophobic loop in the toxin's cell binding domain HC, which is located between the ganglioside- and protein receptor-binding sites, and that is particularly pronounced in the serotypes BoNT/B, DC, and G sharing synaptotagmin as protein receptor. Here, we provide evidence that this HC loop is a critical component of their tripartite receptor recognition complex. Binding to nanodisc-embedded receptors and toxicity were virtually abolished in BoNT mutants lacking residues at the tip of the HC loop. Surface plasmon resonance experiments revealed that only insertion of the HC loop into the lipid-bilayer compensates for the entropic penalty inflicted by the dual-receptor binding. Our results represent a new paradigm of how BoNT/B, DC, and G employ ternary interactions with a protein, ganglioside, and lipids to mediate their extraordinary neurotoxicity.

Biomimetic biosensor to distinguish between inhibitory and non-inhibitory factor VIII antibodies.

Patients with hereditary or acquired haemophilia A may develop inhibitory factor VIII (FVIII) antibodies. These disrupt FVIII activity predominantly by preventing the formation of the tenase complex, leading to a serious bleeding disorder. Antibodies without inhibiting activity, however, can also be found when screening patients with haemophilia A under FVIII supplementation. Therefore, the detection of only these allo- or autoantibodies from plasma is not sufficient. Rather, the characterization of the antibody-induced effects on the coagulation cascade should be considered due to its great diagnostic importance. Currently, inhibitory activities are detected by the functional Bethesda assay, which directly measures the delay in clotting time by the patient plasma. However, this assay does not provide information on the cause of the inhibition. Here, we report the development of a surface plasmon resonance (SPR) biosensor that has the potential to integrate both quantitative and functional information on patient antibody characteristics in one measurement. Recombinant FVIII protein was immobilized on the sensor surface to detect antibodies from patient plasma. The interaction of the FIX- and FXa-clotting proteins with the formed anti-FVIII/FVIII complex could be detected subsequently within the same SPR measurement cycle. Inhibitory antibodies led to the prevention of these interactions. Thus, discrimination between the clinically relevant inhibitory and non-inhibitory antibodies was enabled. In a group of 16 patients with inhibitory antibodies (both ELISA- and Bethesda-positive), 5 patients with non-inhibitory antibodies (ELISA-positive but Bethesda-negative) and 12 healthy controls, diagnostic sensitivity and specificity data of 100% for the FIX interaction were achieved using this biomimetic biosensor approach. The new method allows for detection and quantification, as well as for evaluation of inhibitory activity of allo- and autoantibodies, using small sample volume and short analysis time.

A robust sensor platform for label-free detection of anti-Salmonella antibodies using undiluted animal sera.

We present a portable and easy-to-use biosensor platform, allowing for label-free detection of diagnostic markers in undiluted animal serum. Exemplarily, this is shown for the detection of anti-Salmonella antibodies. 1-lambda-Reflectometry was used as detection method, making the new biosensor platform portable, cheap, and robust. As recognition elements, lipopolysaccharides (LPSs) from Salmonella typhimurium bacteria were immobilized as sensitive layer on the transducer to carry out serological tests via a direct assay format. For this purpose, a new surface preparation protocol has been worked out allowing for immobilization of the LPS via hydrophobic interactions. It has been shown that results obtained by 1-lambda-Reflectometry are equivalent to those obtained by the non-portable Reflectometric Interference Spectroscopy setup. The new sensor platform was calibrated in both matrices, buffer and undiluted serum. Good sensitivity, selectivity and intra chip reproducibility have been observed. Furthermore, inter chip reproducibility was examined and recovery rates were found to be between 99 and 117% in undiluted serum.

A biomimetic sensor surface to detect anti-ß2-glycoprotein-I antibodies as a marker for antiphospholipid syndrome.

A biomimetic sensor has been developed, that allows for quantification of autoantibodies related to the antiphospholipid syndrome (APS). Autoantibodies directed against the ß(2)-glycoprotein-I (ß(2)GP-I) are known as the best markers for diagnosis of APS, however, detection of such antibodies is still a challenge. The epitopes of ß(2)GP-I are exposed upon binding to negatively charged membranes. The surface of the sensor chips was therefore modified with such type of membranes, on which ß(2)GP-I molecules were subsequently immobilized as recognition elements. Using the label-free method, reflectometric interference spectroscopy, it was possible to quantify anti-ß(2)GP-I antibodies and to calibrate the sensor chip in buffer. A mild regeneration procedure allows for many consecutive measurements without stripping off the membrane in between.

A novel analytical tool for quantification of estrogenicity in river water based on fluorescence labelled estrogen receptor alpha.

A novel combined procedure for estrogen-affinity purification and labelling of estrogen receptor alpha ligand-binding domain with Cy 5.5 cystein reactive dye was established. By using this procedure, mainly functional proteins are recovered. It can be easily adapted to a large variety of other proteins for which ligand-coated affinity materials are available. The labelled receptor was used in a total internal reflection fluorescence-based binding inhibition assay for determination of the impact of pollutants in river water on the receptor. The great advantage compared to conventional methods is that the total effect on the receptor is measured instead of concentrations of single compounds and that even currently unknown ligands are found as well. Therefore, the obtained signal is related to the response of the organism, which is exposed to the water. The limit of detection was found to be 0.139 nM of estradiol equivalents. The assay also provides a highly sensitive tool for pharmaceutical research and can be adapted to diagnostic applications.