Protein-sensing assay formats and devices.
Proteins are used as biocatalysts, therapeutic or diagnostic agents, and as such they are biotechnological products. Moreover, they are biomarkers for health states, diseases or toxic or other adverse effects, and the intracellular protein network is essential for the adaptation of an organism to its environment. Thus, there is a strong need for analytical methods for protein determination, which allow not only to indicate the presence of a protein, but also its concentration, covalent modification and activity, and corresponding developments of new methods experienced strong support. Among those methods only those were considered here, which are based on affinity reactions between an immobilized capture agent, such as an antibody or a receptor, and the target protein. Immobilization methods range from adsorption on hydrophobic materials, in membranes or gels to covalent binding and bioaffinity reactions, such as the oriented immobilization of antibodies on protein A/G layers. The applicability of the various methods is dependent on physical and chemical properties of the immobilization substrate and of the capture agent, i.e. the presence of surface charges, hydrophobic areas or functional groups for chemical coupling. The choice of the immobilization substrate is influenced by the combination of the assay and detection principle, which meets best the practical requirements. Assay formats range from direct, label-free one-step detection of the affinity reaction between the capture agent and the target protein to multi-step procedures, such as an enzyme-tracer-based sandwich assays. Each approach has its particular advantages and disadvantages with respect to the complexity of the assay, i.e. number of required reagents and of incubation steps, the possible degree of automation, assay time, availability of suitable reagents, required sample volume, sensitivity and specificity, including the possibility to determine several proteins simultaneously. No general recommendation for the "best choice" was given in this contribution, but examples were chosen, which illustrate the potential of the different systems.