The high specificity and affinity of monoclonal antibodies make them attractive as therapeutic agents. In general, the affinities of antibodies reported to be high affinity are in the high picomolar to low nanomolar range and have been affinity matured in vitro. It has been proposed that there is an in vivo affinity ceiling at 100 pM and that B cells producing antibodies with affinities for antigen above the estimated ceiling would have no selective advantage in antigen-induced affinity maturation during normal immune responses. Using a transgenic mouse producing fully human antibodies, we have routinely generated antibodies with sub-nanomolar affinities, have frequently rescued antibodies with less than 10 pM affinity, and now describe the existence of an in vivo generated anti-hIL-8 antibody with a sub-picomolar equilibrium dissociation constant. This confirms the prediction that antibodies with affinities beyond the proposed affinity ceiling can be generated in vivo. We also describe the technical challenges of determining such high affinities. To further understand the importance of affinity for therapy, we have constructed a mathematical model to predict the relationship between the affinity of an antibody and its in vivo potency using IL-8 as a model antigen. (c) 2005 Elsevier Inc. All rights reserved.