Search

A rapid, label-free optical biosensor system for sensitive monitoring of bio-molecular interactions in real-time is presented. SpectroSens ™ sensor chips are based on integrated planar Bragg gratings sensitive to localised changes in refractive index. Bio-molecule recognition is imparted by functionalisation of the sensing surface with antibodies against targets of interest. In this study, antibodies against selected proteins were oriented with recombinant Protein A/G, which was covalently immobilised to the sensor chip via amine coupling to a glutaraldehyde-activated silane layer. Immunoassays for the detection of rabbit IgG and ovalbumin proteins as model antibody–antigen interaction systems were performed. Binding of complementary antigens to respective antibody-functionalised sensors manifested as changes in wavelength of light reflected from the optical sensors. Quantitative binding kinetics with detection sensitivities in the mid ng/ml range were obtained for both antigens using this planar, two-dimensional surface coating. Data presented demonstrate the suitability of SpectroSens ™ sensors as a valuable tool in life science research and development for monitoring bio-specific interactions, protein concentration determination and antibody selection; the optical integration and analytical characteristics of these sensors suggest that they may find numerous applications in bio-pharmaceutical development and clinical diagnostics.

A reliable ELISA based method has been developed for measuring in solution antigen/antibody association rate constants. Its rationale is as follows; antigen and antibody are mixed in solution to initiate the association. At different time intervals aliquots are withdrawn to determine by an indirect ELISA the amount of free antibody that remains in solution. The disappearance of the free mAb reflects the time course of the association reaction. To test the validity of this method, the association rate constant of a monoclonal antibody for its antigen was measured and compared with that obtained previously by a method using fluorescence. The good agreement between the results obtained with the ELISA-based method and those obtained previously by fluorescence measurement indicates that the method described permits determination of true association rate constants in solution. The present method offers several advantages. It uses only minute amounts of sample which need not be purified; it requires no radioactive or fluorescent labelling of the antibody or the antigen, and it can be applied to any type of complex between macromolecules if an ELISA test can be set up to detect quantitatively one of the macromolecules.

We illustrate use of a simple approach to describe the equilibrium interactions of mixtures of monoclonal antibodies and antigens. This procedure is based on elementary concepts in probability theory and is readily suited to describing interactions of antibodies and antigens which form circular as well as linear complexes. The method is also suited to describing the inhibitory effects of antibodies which compete for overlapping epitopes and an example is provided to show how the procedure can be used to describe the interactions of antibodies which inhibit circular complex formation. We also outline simple strategies for preparing computer programs to simulate binding of antigens to defined antibody mixtures. The methods described should facilitate design of immunoassay procedures based on the use of defined mixtures of monoclonal antibodies.