Ratios of anion and di-anion states of fluorescein (FLU(-1) and FLU(-2)) are 21.2% and 78.8%, respectively, in the neutral pH. We investigated the influence of ionization states of antigen on anti-fluorescein antibodies. For this purpose, steered molecular dynamics (SMD) simulations were performed. Potential of mean forces (PMF) based on Jarzynski equality showed that wild-type (4-4-20) more strongly binds to FLU(-1) than FLU(-2), whereas its femtomolar-affinity mutant (4M5.3) more strongly binds to FLU(-2) than FLU(-1). It was speculated that the environment or the process of in vivo antibody production had been different from those of the protein engineering. (c) 2012 Elsevier B.V. All rights reserved.