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Effects of Hydrophilization and Immobilization on the Interfacial Behavior of Immunoglobulins
The adsorption and immobilization of rabbit anti-human immunoglobulin (rabbit IgG), as well as the effects of rinsing with buffer and addition of bovine serum albumin (BSA) or human IgG on the amount and reactivity of bound rabbit IgG, were investigated with ellipsometry, total internal reflection fluorescence spectroscopy (TIRF), and enzyme immuno assay (EIA). It was found that although rabbit IgG readily adsorbs at hydrophobic hexamethyldisiloxane (HMDSO) plasma polymer surfaces, a substantial fraction of the adsorbed protein molecules is desorbed upon rinsing with buffer. BSA was found to adsorb readily at the surfaces obtained after rinsing, although also this protein desorbed to a large extent (about 60%) upon further rinsing with buffer. The adsorption of BSA causes a further reduction in the amount of rabbit IgG adsorbed. Immobilization of rabbit IgG to acrylic acid (AA) plasma polymer surfaces, achieved by covalent coupling via a strongly adsorbed PEG-PEI copolymer, was found to overcome the problem of the desorption of rabbit IgG upon rinsing with buffer or addition of BSA. Furthermore, nonspecific adsorption was virtually absent after immobilization. However, covalently bound rabbit IgG reacted strongly with human IgG, as observed by ellipsometry, TIRF, and EIA. The immobilization of rabbit IgG to hydrophilized surfaces was found to facilitate the interpretation of EIA results.
The adsorption and immobilization of rabbit anti-human immunoglobulin (rabbit IgG), as well as the effects of rinsing with buffer and addition of bovine serum albumin (BSA) or human IgG on the amount and reactivity of bound rabbit IgG, were investigated with ellipsometry, total internal reflection fluorescence spectroscopy (TIRF), and enzyme immuno assay (EIA). It was found that although rabbit IgG readily adsorbs at hydrophobic hexamethyldisiloxane (HMDSO) plasma polymer surfaces, a substantial fraction of the adsorbed protein molecules is desorbed upon rinsing with buffer. BSA was found to adsorb readily at the surfaces obtained after rinsing, although also this protein desorbed to a large extent (about 60%) upon further rinsing with buffer. The adsorption of BSA causes a further reduction in the amount of rabbit IgG adsorbed. Immobilization of rabbit IgG to acrylic acid (AA) plasma polymer surfaces, achieved by covalent coupling via a strongly adsorbed PEG-PEI copolymer, was found to overcome the problem of the desorption of rabbit IgG upon rinsing with buffer or addition of BSA. Furthermore, nonspecific adsorption was virtually absent after immobilization. However, covalently bound rabbit IgG reacted strongly with human IgG, as observed by ellipsometry, TIRF, and EIA. The immobilization of rabbit IgG to hydrophilized surfaces was found to facilitate the interpretation of EIA results.