Identification and optimization of regeneration conditions for affinity-based biosensor assays. A multivariate cocktail approach

A general regeneration, identification, and optimization (RO) protocol for Biacore systems was developed. The RO protocol uses six multi-ingredient st ock solutions that represent the six most common chemical properties employ ed as regeneration agents. The regeneration effect of different regeneratio n cocktails of these six stock solutions were tested iteratively until a sa tisfactory result was obtained. The RO protocol was designed with an ease-o f-use and multivariate approach. The RO protocol was tested on 13 different antibody-antigen systems. For 10 of these, only the first screening sessio n was tested. For 9 of the 13 systems, the RO-protocol screening session id entified cocktails that removed more than 90% of the bound analyte in a 30 s pulse. For 5 systems, the RO protocol identified cocktails that regenerat ed the surface completely and that were more gentle than previously used re generation conditions. Furthermore, the regeneration optimization results c an be interpreted as a characterization of the interacting molecules. The r elevance of testing cocktails was justified by the fact that at least one c ocktail was significantly better than all diluted stock solutions for all t ested model systems. By using the multivariate approach, the risk of missin g relevant combinations of stock solutions was minimized. This resulted in an unexpected discovery of excellent properties of EDTA. as an additive in regeneration cocktails containing chaotropic agents and ions in high concen tration.