RESUMO
An optical biosensor system employing a resonant mirror (RM), with a stirred cuvette has been used to follow the interaction of a recombinant antibody fragment with its antigen, hen egg lysozyme. The data generated by the biosensor were analysed in order to determine the kinetic constants for the interaction using a linear transform (derivative analysis). For comparison the data were also analysed using an exponential curve fitting routine. It was demonstrated that the exponential curve fitting method produced results which were in agreement with the existing linear transform method. It was also shown that early fitting of the association phase response, using the exponential curve fitting routine between 0 and 70 s after sample addition, yielded sufficient information to provide a prediction of Kon. The potential use of the optical biosensor for the rapid monitoring of protein production and purification is discussed.
Assuntos
Reações Antígeno-Anticorpo/fisiologia , Técnicas Biossensoriais , Óptica e Fotônica/instrumentação , Animais , Anticorpos/genética , Biotecnologia , Galinhas , Interpretação Estatística de Dados , Enzimas Imobilizadas , Feminino , Cinética , Muramidase/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Methods based on the use of optical biosensors have recently become available to provide a convenient means of determining the rate and equilibrium constants for bimolecular interactions between immobilized ligands and soluble ligate molecules. However, the association data that these methods provide are not always accurately described by the expected pseudo-first-order reaction mechanism, particularly when the ligand is immobilized on a dextran matrix. We show that a better description of the association data, especially at higher ligate concentrations, is achieved with a double exponential function, indicating that at least two rate-limiting processes are involved. Various models are considered in order to explain these observations: the presence of two (or more) distinct populations of immobilized ligand; a change, possibly conformational, in the immobilized ligand before or after ligate binding; or the hindrance of ligate binding to immobilized ligand. We suggest that steric hindrance caused by ligate binding to the dextran-coated sensor surface seems the most likely explanation for the observed biphasic association kinetics and that the faster initial phase should be used in oder to determine association constants that can be compared to those in solution.
Assuntos
Proteínas/metabolismo , Reações Antígeno-Anticorpo , Técnicas Biossensoriais , Cinética , Modelos Teóricos , Ligação ProteicaRESUMO
The potential of a new optical biosensor, the resonant mirror, for detecting whole cells is demonstrated. Staphylococcus aureus (Cowan-1) cells, which express protein-A at their surface, were detected by binding to human immunoglobulin G (IgG) immobilized on an aminosilane-derivatized sensor surface at concentrations in the range 8 x 10(6)-8 x 10(7) cells/mL. A control S. aureus strain (Wood-46), which does not express protein-A, gave no significant response. Immobilization of the capture ligand on aminosilane surfaces with and without a hydrogel coating of carboxymethyl-dextran was compared. The greatest binding response was observed with non-dextran-coated surfaces. The sensitivity of the technique was increased a 1000-fold by using a human IgG-colloidal gold conjugate (30 nm) in a sandwich assay format. S. aureus (Cowan-1) cells were detected in spiked milk samples at cell concentrations from 4 x 10(3)-1.6 x 10(6) cells/mL using the sandwich assay.
