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Rebinding of dissociated ligands from cell surface proteins can confound quantitative measurements of dissociation rates important for characterizing the affinity of binding interactions. This can be true also for in vitro techniques such as surface plasmon resonance (SPR). We present experimental results using SPR for the interaction of insulin-like growth factor-I (IGF-I) with one of its binding proteins, IGF binding protein-3 (IGFBP-3), and show that the dissociation, even with the addition of soluble heparin in the dissociation phase, does not exhibit the expected exponential decay characteristic of a 1:1 binding reaction. We thus consider the effect of (multiple) rebinding events and, within a self-consistent mean-field approximation, we derive the complete mathematical form for the fraction of bound ligands as a function of time. We show that, except for very low association rate and surface coverage, this function is non-exponential at all times, indicating that multiple rebinding events strongly influence dissociation even at early times. We compare the mean-field results with numerical simulations and find good agreement, although deviations are measurable in certain cases. Our analysis of the IGF-I-IGFBP-3 data indicates that rebinding is prominent for this system and that the theoretical predictions fit the experimental data well. Our results provide a means for analyzing SPR biosensor data where rebinding is problematic and a methodology to do so is presented.1 Present Address: Max Planck Institut für Physik komplexer Systeme, Nöthnitzer Stra e 38, 01187 Dresden, Germany. (i) IntroductionSignal transduction via transmembrane receptor proteins is initiated by extracellular binding with specific proteins known as growth factors. These interactions tend to be of high affinity and, in many systems, are regulated by binding proteins present in the extracellular environment. Insulin-like growth factor-I (IGF-I) constitutes one prominent example of such a growth factor. Cell signaling is transmitted by direct interaction with the IGF-I receptor but this binding can be impacted by solution and cell-associated IGF binding proteins (IGFBPs), of which there are at least six. Quantification of the interactions of IGF-I with IGFBPs is critical if one is to understand how changes in expression and secretion will impact IGF-I signaling. Surface plasmon resonance (SPR) is one technique amenable to such measurements. SPR is an optical sensor technique that has the advantage of being able to take real-time measurements using low concentrations of unlabeled biologicals [reviewed in Cooper 2003].Quantification of IGF-I interactions with both cell surface receptors and IGFBPs using SPR has been performed as a means of evaluating and predicting the competition between these molecules for IGF-I. Studies have used immobilized IGF-I [Wong et al. 1999;Dubaquie and Lowman 1999;Galanis et al. 2001;Fong et al. 2002; Vorwerk et al. 2002], IGF-I receptor [Jansson et al. 1997], or IGFBPs [Heding et al. 1996Jansson et al. ...

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To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.