Total internal reflection fluorescence with energy transfer: a method for analyzing IgG adsorption on nylon thin films

Abstract

Total internal reflection fluorescence was used to analyze rabbit immunoglobulin G (IgG) nonspecifically adsorbed on thin nylon f i i . Fluorescence energy transfer measurements were recorded for two comparative systems: (1) adsorbed fluorescein isothiocyanate (FITC) tagged IgG and tetramethylrhodamine isothiocyanate (TRITC) tagged nylon; (2) FITC labeled IgG and TRITC labeled anti-rabbit IgG (anti-IgG). These experiments were used to examine the nylon/IgG and IgG/solution interfaces, respectively, as a function of the surrounding buffer pH and washing conditions. Results were correlated with IgG mass loading measured by the bicinchoninic acid (BCA) method and anti-IgG binding activity assessed using TRITC-tagged anti-IgG. When washed with pH 6.9 phosphate buffer, the amount of adsorbed IgG increased with the concentration of IgG used in the adsorption step. Fluorescence data were consistent with an IgG bilayer model, where the upper layer did not interact with the TRITC labeled nylon surface but bound to and exchanged with solution borne anti-IgG. After washing with carbonate buffer at pH 8.7, only a tightly adsorbed, unexchangeable layer of IgG remained on the nylon surface, with mass corresponding to less than a monolayer. In a pH 8.7 buffer, no energy transfer took place between adsorbed FITC labeled IgG and the TRITC tagged anti-IgG although anti-IgG did adhere to the nylon, suggesting coadsorption of anti-IgG rather than immunochemical binding. When the buffer was replaced by a pH 6.9 phosphate buffer, energy transfer was observed, indicating that binding had occurred.

Effects of Discrete Protein-Surface Interactions in Scanning Force Microscopy Adhesion Force Measurements

“…16 The SFM topographic Figure 2 shows typical results from a series of SFM adhesion measurements taken using the molecularly specific system, i.e. a fluoresceinated SFM probe and anti-fluorescyl IgG surface.…”

Feasibility of measuring antigen–antibody interaction forces using a scanning force microscope

“…Immobilization of 125 I-labeled antifluorescyl IgG to a GPS surface showed a protein surface density of 0.5 μg cm −2 , or one molecule per 40 nm 2 . This corresponds to slightly less than a monolayer, which has been determined to be between 0.6 and 2.1 μg cm −2 for IgG, depending on the molecular orientation [33].…”

Adsorption of Monoclonal Antibodies to Glass Microparticles

“…A number of published reports discuss the adsorption capacity of a wide range of different materials for antibodies and other proteins in solutions which largely approximate physiological conditions found in serum 21–29. In contrast, we examine humAb interactions with glass microparticles in solution conditions typically used for therapeutic antibody formulations (e.g., pH 6, low ionic strength).…”