Quantification of transport and binding parameters using fluorescence recovery after photobleaching. Potential for in vivo applications

Abstract

Fluorescence Recovery After Photobleaching (FRAP) has been used extensively in the study of transport and binding in biological media in vitro. The present study adapts and further develops FRAP so that it may be utilized for the in vivo quantification of binding parameters. The technique is validated in vitro by measuring mass transport and binding parameters for the Concanavalin A/Mannose binding system (a diffusion-limited system). The pseudo-equilibrium constant (the product of the equilibrium constant and the total concentration of binding sites) for this system was determined to be 26 +/- 15 which compares favorably with literature values ranging between 16 and 32. The applicability of this technique to measure parameters for monoclonal antibody/antigen interactions in a thin tissue preparation such as the rabbit ear chamber tissue preparation is also examined. Unlike other methods for measuring binding parameters, this is the only technique which has the potential to measure parameters relevant to antibody delivery in vivo. The proposed technique is noninvasive and does not require a priori knowledge of, independent measurement of, or variation in the concentration of binding sites or total concentration of binding species.

Rapid Glucocorticoid Receptor Exchange at a Promoter Is Coupled to Transcription and Regulated by Chaperones and Proteasomes

“…The FRAP data are given by frap(t) ϭ e Ϫ2r/t [I 0 (2r/t) ϩ I 1 (2r/t)], where t is time and I 0 and I 1 are modified Bessel functions. Bessel functions and their variants typically arise in differential equations with cylindrical symmetry (1), as occurs for a FRAP with a circular bleach spot (15). The Soumpasis theory presumes a normalized FRAP that ranges from 0 to 1.…”

“…When diffusion and binding interactions are present, the simplest scenario is effective diffusion (15). In effective diffusion, the FRAP mimics diffusion but at a lower rate given by the equation D eff ϭ D/͑1 ϩ k * on /k off ͒, where D is the cellular diffusion constant, k off is the off rate of binding, and k * on is the product of the on rate for binding times the equilibrium concentration of binding sites.…”

“…The simplest scenario to explain combined diffusion and binding is effective diffusion (15). Here, the FRAP exhibits a slowed diffusion, with the "effective" diffusion constant retarded in proportion to the binding affinity.…”

“…Finally, in its simplest form, the effective-diffusion theory predicts a pseudoequilibrium binding constant (15), but only for the case where the distribution of fluorescence is homogeneous. This should apply to generic locations in the nucleus, but at present such estimates cannot be obtained for the array because the theory must be extended to account for such a nonhomogeneous fluorescence distribution in which a bright region (the array) is surrounded by a dimmer region (the rest of the nucleus).…”

Directin vivomeasurement of targeted binding in a human tumor xenograft

“…This approach has been used extensively to detect binding in cell membranes (7,8), cytoplasm (9,10), and various in vitro preparations (11)(12)(13)(14)(15)(16). We previously applied FRAP in vivo to measure the interstitial diffusion and convection of albumin within a tumor tissue preparation (17), and we report here the application of FRAP to measure binding in vivo.…”