TOTAL INTERNAL-REFLECTION FLUORESCENCE WITH ENERGY-TRANSFER - A METHOD FOR ANALYZING IGG ADSORPTION ON NYLON THIN-FILMS

Total internal reflection fluorescence was used to analyze rabbit immunoglobulin G (IgG) nonspecifically adsorbed on thin nylon films. 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.