RESUMO
Biolayer interferometry is a novel method for quantifying macromolecules, such as proteins, in solution. The presence of other, non-binding molecules does not interfere with quantification, which allows one to measure the concentration of the molecule of interest in a crude mixture. Here we apply this method to determining the dynamic binding capacity of affinity resins.
Assuntos
Proteínas/isolamento & purificação , Animais , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Ligação Proteica , Proteínas/metabolismoRESUMO
The focus in the field of protein arrays has shifted from technology development to applying the technology in biological research. This review will highlight several recently published examples of biologically relevant experiments using both planar and bead-based arrays. Examples of the use of antibody arrays, antigen arrays and protein activity arrays will be discussed.:
RESUMO
The analysis of biomolecular interactions is key in the drug development process. Label-free biosensor methods provide information on binding, kinetics, concentration, and the affinity of an interaction. These techniques provide real-time monitoring of interactions between an immobilized ligand (such as a receptor) to an analyte in solution without the use of labels. Advances in biosensor design and detection using BioLayer Interferometry (BLI) provide a simple platform that enables label-free monitoring of biomolecular interactions without the use of flow cells. We review the applications of BLI in a wide variety of research and development environments for quantifying antibodies and proteins and measuring kinetics parameters.
Assuntos
Técnicas Biossensoriais/métodos , Interferometria/métodos , Anticorpos/química , Reações Antígeno-Anticorpo , Técnicas Biossensoriais/instrumentação , Descoberta de Drogas/métodos , Cinética , Ligantes , Proteínas/química , Coloração e Rotulagem , Fatores de TempoRESUMO
Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function of the surface density and fractional activity of the capture agents. Here we investigate how these two factors are affected by the orientation of the capture agents on the surface. We compare randomly versus specifically oriented capture agents based on both full-sized antibodies and Fab' fragments. Each comparison was performed using three different antibodies and two types of streptavidin-coated monolayer surfaces. The specific orientation of capture agents consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvements over surfaces with randomly oriented capture agents. Surface plasmon resonance revealed a dense monolayer of Fab' fragments that are on average 90% active when specifically oriented. Randomly attached Fab's could not be packed at such a high density and generally also had a lower specific activity. These results emphasize the importance of attaching proteins to surfaces such that their binding sites are oriented toward the solution phase.