ABSTRACT
Molecular and functional analysis of small molecule binding to protein can provoke insights into cellular signaling and regulatory systems as well as facilitate pharmaceutical drug discovery. In label free small molecule detection the displacement assay format can be applied. This is beneficial because displacement of high molecular weight receptors is detected instead of low molecular weight ligand as in classical binding analysis. Thus, detection limit is potentially lowered. Using the influenza haemagglutinin (HA) peptide binding to mono or bivalent anti-haemagglutinin peptide antibody displacement assay formats could be established. The exact time resolved analysis of binding and dissolution of ligand HA and anti-Haemagglutinin peptide antibody was achieved with surface plasmon resonance (SPR) spectroscopy. Mathematical models could be developed from kinetic equations of ligand binding to mono or bivalent antibodies. With this, an accurate simulation of the SPR results was reached. The simulation plot had to be exactly adjusted to the SPR results to determine all kinetic rate constants defining ligand and receptor binding kinetics. Large variations in receptor concentration gave almost identical rate constants in binding. It became obvious that rebinding is in any case not necessary to understand the binding kinetics of our model system HA/anti-HA. Maximum decline of SPR response could be used to determine ligand concentrations in analyte.
Subject(s)
Antibodies, Bispecific/chemistry , Antibodies, Monoclonal, Murine-Derived/chemistry , Antibodies, Viral/chemistry , Antigen-Antibody Complex/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Surface Plasmon Resonance/methods , Animals , Antibodies, Bispecific/immunology , Antibodies, Monoclonal, Murine-Derived/immunology , Antibodies, Viral/immunology , Antigen-Antibody Complex/immunology , Antigen-Antibody Reactions , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Mice , Models, ChemicalABSTRACT
Solidly mounted (SMR-type) thin film bulk acoustic resonators operating at 2.2, 4.1, and 8.0 GHz and with lateral extents from 30 to 500 microm were fabricated and their performance as mass sensors was evaluated theoretically as well as experimentally. It was found that increasing the frequency leads to a principally improved performance of these devices. Problems arising for the horizontal as well as the vertical dimension and structure are investigated.
