FINISHED PROJECT: Appropriate design of microspot immunoassays; compensation for kinetic limitations                                       We examined the limitations of existing microarray immunoassays and investigated how best to optimise them using theoretical and experimental approaches. A key physicochemical limitation of microarray immunoassays is a strong dependence of antibody microspot kinetics on the mass flux to the spots. We analysed theoretically and experimentally the effects of microarray design parameters (incubation vessel geometry, incubation time, stirring, spot size, antibody-binding site density, etc.) on microspot reaction kinetics and sensitivity. Using a two-compartment model, the quantitative descriptors of the microspot reaction were determined for different incubation and microarray design conditions. This analysis revealed profound mass transport limitations in the observed kinetics, which may be slowed down as much as hundreds of times compared with solution kinetics. The data obtained were considered with relevance to microspot assay diffusional and adsorptive processes, enabling us to validate some of the underlying principles of the antibody microspot reaction mechanism and provide guidelines for optimal microspot immunoassay design. For assays optimised to maximize the reaction velocity, we could demonstrate sensitivities in the attomolar and low femtomolar ranges.

Kusnezow et al. (2005) Handbook of Immunohistochemistry, Vol. 2 (Hayat, M.A., ed.), Elsevier, 23-35. Klenin et al. (2005) J. Chem. Phys. 122, Art. No. 214715. Syagailo et al. (2006) Apoptosis and Cancer Therapy (Debatin, K.M. & Fulda, S., eds.), Wiley-VHC, 60-85. Kusnezow et al. (2006) Proteomics 6, 794-803. Kusnezow et al. (2006) Expert Rev. Mol. Diagn. 6, 111-124. Kusnezow et al. (2006) Mol. Cell. Prot. 5, 1681-1696.

FINISHED PROJECT: Evaluation of labelling strategies                                    The suitability of simple and inexpensive detection approaches for highly sensitive antibody microarray analysis was examined. N-hydroxysuccinimidyl ester (NHS) and Universal Linkage System (ULS) based fluorescein and biotin labels used as tags for subsequent detection with anti-fluorescein and extravidin, respectively, as well as fluorescent dyes were applied for analysis of blood plasma. Parameters modifying strongly the performance of microarray detection such as labelling conditions, incubation time, concentrations of anti-fluorescein and extravidin and extent of protein labelling were analysed and optimised. Indirect detection strategies whether based on NHS- or ULS-chemistries strongly outperformed direct fluorescent labelling and enabled detection of low abundant cytokines with many dozen-fold signal-to-noise ratios. Kusnezow et al. (2007) Proteomics 7, 1786-1799.

FINISHED PROJECT: .Subcellular protein extraction from human pancreatic cancer tissue                  Proteins are the major class of effector molecules in cellular systems. For the identification of functional differences between normal and diseased tissues, a reliable analysis of their protein content is essential. Reproducible isolation and fractionation of intact proteins are important in this respect. The complexity of proteins in structure and concentration, their close interaction as well as their instability represent major challenges. For isolation from tissues, also the destruction of the cell-cell and cell-matrix connections within a tissue without affecting protein quality is a critical factor. We compared different processes for a compartmental protein preparation from pancreatic tissues, using matching normal and tumour tissue samples from the same patients. Pancreas is one of the most challenging tissues because of its high content of proteases. .. Success of the different procedures varied strongly. Based on a scheme of slicing the tissues and a subsequent isolation of the cells, we established a workflow for the extraction in a reproducible manner of fractions of cytosolic proteins, membrane and organelle proteins, nuclear proteins and cytoskelatal filaments. The tissue slices also allow for a representative confirmation of the individual samples’ cellular status by histochemical processes and a proper separation or mixing of cellular material from across a tumour if required. Börner et al. (2009) BioTechniques 46, 297-304.