Scientific projects are driven by the delivery of quality analytical data at the right time. The lack of robust analytical methods frequently hinders early progress due to the historical lag time that exists between project conception and assay development. For example, modern cell-line development methodologies are able to transition from transfection to selection in 30 days, allowing just a few weeks for development of a quantitation screening assay for clone selection.
While this lack of synchronicity cannot be eliminated, its minimization through advancements in analytical technologies represents an opportunity for increased efficiency, speed, and dexterity. This tutorial describes the rapid evaluation (15 minutes) of biosensor chemistries for a quantitation assay on the ForteBio Octet instrument using a multi-analyte workflow and demonstrating an accelerated pace of assay development.
ForteBio’s Octet systems offer a dip-and-read platform that provides easy, fast, and high-quality quantitation and kinetic data in a 96- or 384-well plate where existing methods have limitations in throughput, performance, and cost. The Octet instrument can read 16 quantitation samples in as little as 2 minutes and 384 quantitation samples in as little as 60 minutes, accelerating both assay development and delivery of decision-making data.
The systems utilize a BioLayer Interferometry (BLI)-based technology to detect molecular interactions on the tip of a fiber optic biosensor. BLI is an optical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip and an internal reference layer.
The instrument may be used for quantitation by measuring the rate of association of an analyte, such as a human IgG1 with a ligand immobilized on the biosensor, such as protein A. Higher antibody concentrations cause faster binding rates and analyte concentration is calculated from the binding rate using a standard curve. With highly specific ligands, such as the immunoglobulin-binding protein A, biosensors can directly quantitate antibody analytes in conditioned media and complex matrices.
When developing a quantitation assay on the Octet platform, the first question is, “which biosensor chemistry should I use?” ForteBio currently offers six out-of-the-box biosensors for quantitation: protein A, protein G, protein L, anti-human IgG Fc, antimurine IgG Fv, and anti-penta-His. As new biosensor chemistries are regularly developed, refer to www.fortebio.com/bio?sensor_types.html for the most current list. Custom quantitation biosensors can be developed using streptavidin and amine reactive biosensors.
By employing a multi-analyte workflow, this experiment evaluates the potential use of proteins A, G, and L biosensors for two antibody analytes using the Octet platform. Proteins A, G, and L are immunoglobulin-binding proteins originally sourced from microorganisms and have been widely adapted as protein analytical tools due to their high affinity and selectivity.
Proteins A and G bind to the Fc domain of antibodies while protein L binds to the kappa light chain. Proteins A, G, and L do not bind universally to all immunoglobulins and tables have been prepared as guidelines for their specificity. However, because each antibody possesses a unique set of binding properties, and because sometimes the isotype of the antibody is unknown, experimental evaluation against a target analyte is the most definitive means of choosing a biosensor chemistry for subsequent assay development.