In situ FTIR ATR spectroscopic study of the interaction of immobilized human tumor necrosis factor-alpha with a monoclonal antibody in aqueous environment.
By in situ FTIR ATR measurements, the antibody (AB) recognition of human tumor necrosis factor-alpha (TNFalpha) immobilized on the Ge surface of a multiple internal reflection element (MIRE) was investigated. The experiments were performed in aqueous environment in a flow-through cell. After immobilization of TNFalpha on the Ge-MIRE by direct adsorption from aqueous solution, the immobilisate reached stability after about 1 h under flow-through conditions. The remaining sites of the Ge surface were saturated by bovine serum albumin (BSA) in order to prevent unspecific binding of anti-TNFalpha AB which was then added. The obtained FTIR ATR spectra were shown to result exclusively from AB specifically interacting with TNFalpha, since the absence of immunoglobulin binding to BSA adsorbed to the Ge MIRE was verified by a reference experiment. Finally, the stability of all adsorbed protein immobilisates was monitored under flow-through conditions for 10.5 h. The TNFalpha-AB complex showed a decrease of 7.4%, whereas the BSA adsorbate remained stable. IR measurements were performed with polarized light in order to study orientational effects of the immobilized proteins. The dichroic ratios and surface concentrations of all used proteins are available after quantitative analysis of the amide II bands.
Reiter
Gerald
G
Institute of Physical Chemistry, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria.
Hassler
Norbert
N
Weber
Viktoria
V
Falkenhagen
Dieter
D
Fringeli
Urs Peter
UP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D002417:Cattle; D003879:Dermatologic Agents / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D005857:Germanium / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D000069285:Infliximab; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000502:physiology; D017550:Spectroscopy, Fourier Transform Infrared; D013499:Surface Properties; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003879:Dermatologic Agents; D014409:Tumor Necrosis Factor-alpha; D005857:Germanium; D012710:Serum Albumin, Bovine; D000069285:Infliximab