Label-free assessment of high-affinity antibody-antigen binding constants. Comparison of bioassay, SPR, and PEIA-ellipsometry.

Assessment of high-affinity antibody-antigen binding parameters is important in such diverse areas as selection of therapeutic antibodies, detection of unwanted hormones in cattle and sensitive immunoassays in clinical chemistry. Label-free assessment of binding affinities is often carried out by immobilization of one of the binding partners on a biosensor chip, followed by monitoring the binding equilibrium of the other partner. However, for the measurement of high-affinity binding, with dissociation constants in the picomolar range or lower, equilibration times exceed practical limits and one has to resort to the measurement of sorption kinetics.

The bioanalysis of trastuzumab in human serum using precipitate-enhanced ellipsometry.

For the bioanalysis of therapeutic monoclonal antibodies in biological matrices, immunoassays--especially enzyme-linked immunosorbent assays (ELISAs)--are the most widely used techniques. Although ELISAs are very sensitive, the obtained sensitivity is not always sufficient. In this study, we have investigated the possibilities of performing a precipitate-enhanced immunoassay (PEIA) with ellipsometric detection for the bioanalysis of the therapeutic monoclonal antibody trastuzumab.

Surface-dependent transitions during self-assembly of phospholipid membranes on mica, silica, and glass.

Formation of supported membranes by exposure of solid surfaces to phospholipid vesicles is a much-used technique in membrane research. Freshly cleaved mica, because of its superior flatness, is a preferred support, and we used ellipsometry to study membrane formation kinetics on mica. Neutral dioleoyl-phosphatidylcholine (DOPC) and negatively charged dioleoyl-phosphatidylserine/dioleoyl-phosphatidylcholine (20% DOPS/80% DOPC) vesicles were prepared by sonication.

Effects of flow on solute exchange between fluids and supported biosurfaces.

Uptake of nutrients by cultured cells on solid supports, conversion of substrates by surface-bound catalysts and binding of antibodies to microtitre plates are examples of transport processes that are strongly influenced by the flow conditions in the surrounding fluid. The literature on this subject is scattered over widely different research fields and is often found in dated, and not generally available, treatises. Also, the subject is inherently complicated from a mathematical viewpoint, because even the simplest experimental configurations will usually not allow analytical solutions for the diffusion-convection equations describing the solute mass transport in the system.

One-step immunoassay for measuring protein concentrations in plasma, based on precipitate-enhanced ellipsometry.

Standard enzyme immunoassays (EIAs) require washing steps to remove excess enzyme-antibody complexes. Such washing is laborious, lengthens assay time, and increases assay scatter. Recently, so-called precipitate-enhanced immunoassays (PEIAs) were introduced.

Anticoagulant and membrane-degrading effects of secretory (non-pancreatic) phospholipase A2 are inhibited in plasma.

Plasma concentrations of secretory (non-pancreatic) phospholipase A2 (sPLA2) may rise 1000-fold during inflammation, and this acute phase response has been related to anticoagulant effects. In the present study this hypothesis was further investigated. Prothrombinase activity was measured for model membranes mimicking the phospholipid composition of the outer membrane of resting and activated blood platelets.