Antibody-antigen interactions: what is the required time to equilibrium?. 2010. Available from Nature Precedings

The use of antibodies is widespread in many areas including in-vivo and in-vitro diagnostics, quantitative analysis in research laboratories and as therapeutic substances. Since the methods for generation of antibodies has improved and regularly results in high-affinity interactions, the standard assays used for quantification of the interaction properties should be revisited because they do not necessarily produce accurate results. Here we show that in several cases, the affinity determination of strongly binding antibodies will be inherently difficult when using standard procedures, due to impractically long incubation times. Real-time kinetic analysis is often the only realistic alternative for affinity determination. The estimate of how strongly an antibody binds to its antigen is often reported as the affinity, KD, of the interaction. The affinity can be measured using a variety of assays, most requiring that the reading takes place when equilibrium has occurred. The increased focus on high-affinity antibodies (typically KD <1 nM) has led to a situation where the time to equilibrium increases vastly. The essence of reading at equilibrium is seldom discussed, and the appropriateness of protocols developed for normal affinity antibodie