Kinetics of ligand binding to membrane receptors from equilibrium fluctuation analysis of single binding events.

Equilibrium fluctuation analysis of single binding events has been used to extract binding kinetics of ligand interactions with cell-membrane bound receptors. Time-dependent total internal reflection fluorescence (TIRF) imaging was used to extract residence-time statistics of fluorescently stained liposomes derived directly from cell membranes upon their binding to surface-immobilized antibody fragments. The dissociation rate constants for two pharmaceutical relevant antibodies directed against different B-cell expressed membrane proteins was clearly discriminated, and the affinity of the interaction could be determined by inhibiting the interaction with increasing concentrations of soluble antibodies.

CD4+ T cells have a key instructive role in educating dendritic cells in allergy.

Background: Dendritic cells (DCs) are central in allergy as regulators of the Th1/Th2 balance. We have recently demonstrated a unique transcriptional profile of DCs in patients with ongoing allergy compared with healthy subjects and shown that crosstalk between DCs and memory T cells affects the transcriptional profile of T cells. However, the transcriptional profile of DCs educated by T cells in allergy is unknown.

Augmented Phl p 5-specific Th2 response after exposure of dendritic cells to allergen in complex with specific IgE compared to IgG1 and IgG4.

In this study we have elucidated the effects of allergen-specific antibodies on human DCs and T-cells. Monocyte-derived DCs from allergic patients were exposed to Phl p 5 alone or in complex with Phl p 5-specific human IgG1, IgG4 or IgE and further co-cultured with autologous memory CD4(+) T-cells. We demonstrate that DCs treated with Phl p 5/IgE-complexes secrete higher levels of IL-1alpha, IL-6, VEGF and MCP-3 compared to Phl p 5 alone.

Design of recombinant antibody microarrays for cell surface membrane proteomics.

Generating proteomic maps of membrane proteins, common targets for therapeutic interventions and disease diagnostics, has turned out to be a major challenge. Antibody-based microarrays are among the novel rapidly evolving proteomic technologies that may enable global proteome analysis to be performed. Here, we have designed the first generation of a scaleable human recombinant scFv antibody microarray technology platform for cell surface membrane proteomics as well as glycomics targeting intact cells.

Design of recombinant antibody microarrays for complex proteome analysis: choice of sample labeling-tag and solid support.

Antibody-based microarray is a novel technology with great potential within high-throughput proteomics. The process of designing high-performing antibody (protein) microarrays has, however, turned out to be a challenging process. In this study, we have developed further our human recombinant single-chain variable-fragment (scFv) antibody microarray methodology by addressing two crucial technological issues, choice of sample labeling-tag and solid support.

Design of atto-vial based recombinant antibody arrays combined with a planar wave-guide detection system.

Antibody microarray is a rapidly emerging, powerful approach with great promise within high-throughput proteomics. However, before a truly proteome-wide analysis can be performed, the antibody array format needs to be miniaturized even further in order to enable ultradense arrays to be fabricated. To this end, we have designed and generated proof-of-concept for the first generation of an atto-vial based recombinant antibody array platform.

Evolution of a carbohydrate binding module into a protein-specific binder.

A carbohydrate binding module, CBM4-2, derived from the xylanase (Xyn 10A) of Rhodothermus marinus has been used as a scaffold for molecular diversification. Its binding specificity has been evolved to recognise a quite different target, a human monoclonal IgG4. In order to understand the basis for this drastic change in specificity we have further investigated the target recognition of the IgG4-specific CBMs.