Octet Assay Tips for Biacore Users

Phil Buckle, Applications Manager, Europe
pbuckle@fortebio.co.uk

Common Techniques for Immobilizing the Ligand on Biosensors

Amine Coupling

Still the most common route for covalent ligand immobilization on Biacore, amine coupling uses carboxymethyl dextran-coated chips, which vary in thickness and degree of carboxylation depending on the specific ligand and analyte properties (size, charge, etc).

For immobilizing antibodies to the EDC/NHS activated carboxylic acid surface, pH 5 buffer with an ionic strength around 10 mM is typically used. When transferring amine coupling from Biacore to the Octet, keep in mind that the Octet Amine Reactive biosensors do NOT carry a dextran matrix, are not based on a gold substrate, and have a much lower degree of carboxylation. In practice, this leads to the following considerations:

• MES buffers are preferable for use with Octet Amine Reactive biosensors
• Optimal pH for ligand immobilization on the Octet system may be different from the optimal pH on Biacore
• Amine coupling on the Octet is most effective for ligands with MW >50 kDa
• Recommend starting ligand concentration should be 167 nM or more

As a general strategy, the eight channels on the Octet Q, QK and RED, and 16 channels on the Octet QK384 and RED384 systems should be taken advantage of to simultaneously optimize both ligand loading concentration and buffer pH. This is a rapid parallel method, and conditions can be optimized in the time it takes to run a single immobilization.

Once conditions are optimized for the immobilization, the re-racking feature on the Octet QK384 and RED384 systems can be advantageously utilized to do batch preparation of protein-loaded biosensors by repeated use of a single set of ligand and blocking reagent wells in the sample plate.

Streptavidin/Biotin Capture

This method has the following advantages:

• Biotinylation is performed at neutral pH, circumventing problems with unstable and/or acidic ligands
• By biotin labeling at a molar ratio of 1:1 (biotin:ligand) and with a biotinylation reagent incorporating a long-chain linker, there is less chance for cross-linking and steric hindrance of the ligand once immobilized (as is possible with the standard amine coupling procedure). This will result in more active and homogeneous ligand surfaces.
• Biotin-labeled ligand may be prepared batch-wise and stored for subsequent immobilizations. (See ForteBio Technical Note 10: Batch Immobilization of a Biotinylated Ligand onto Streptavidin Biosensors)
• Typically, the unused biotin labeled ligand can be recovered from the sample plate and reused; alternately, the ligand can be re-used several times to load multiple Streptavidin biosensors for batch preparation on the Octet QK384 and RED384 systems.
• As with amine coupling, the 8 channels on the Octet Q, QK and RED and 16 channels on the Octet QK384 and RED384 systems can be taken advantage of to optimize ligand loading concentration rapidly.

Other Capture Techniques

Other capture techniques are available using the Octet biosensors. These include capture of his-tag ligands, through either biotin-NTA (optimized as above) or amine coupled anti-his antibody. The Octet Aminopropylsilane biosensor (APS) is analogous to the Biacore HPA surface and can be used for immobilizing hydrophobic ligands.

Analyte Binding Experiments

Flow-based systems such as Biacore require that a regeneration protocol be optimized when even a moderately high affinity interaction of ligand (around 50 nM or less) and analyte is being studied. To simplify this, reversible capture techniques may be used, but this is not possible if the ligand is amine-coupled or captured via biotin/streptavidin interaction. Typically, many Biacore chips (and indeed ligand) need to be sacrificed whilst searching for the optimum regeneration buffer, which must completely remove bound analyte and leave the ligand activity unaffected.

By using the processing power of up to 16 biosensors in parallel on the Octet systems, kinetics experiments can be performed without the need for regeneration. This can have great advantages in assay development speed, and, as the Octet system biosensors are disposable, the cost advantages are significant.

Mass transport limitations are an important consideration of kinetics assays in surface-bound techniques such a Biacore and the Octet system. Typically, during assay development on the Biacore, a series of analyte binding experiments are performed where the concentration of analyte is fixed, and the flow rate is varied. If the observed binding rate is not flow dependant, it is assumed that the binding is not mass transport limited. A similar set of experiments can be performed by varying the stir speed during initial experiments on the Octet systems.

Optimization of buffer conditions can be time consuming in flow based systems, since only a limited set of conditions can be addressed at any time. For the reasons mentioned previously, up to 16 buffer conditions can be assessed on the Octet systems simultaneously. Importantly, these conditions can be assessed in the time it takes for a single analyte binding run (indeed, often in the time taken to prime flow based systems from one buffer to another).

Small Molecule Binding Assays

In small molecule assays, solubility issues usually mean that assays are run in a background of DMSO (usually around 5% v/v in running buffer). Even small mismatches of DMSO concentration can give rise to significant signals in SPR-based instruments and must be corrected for by running DMSO correction curves. Octet systems use BLI as the detection principle, which is less sensitive to refractive index changes, and thus, solvents such as DMSO are much less likely to cause problems. Indeed, DMSO mismatches of several percent can be tolerated between samples and running buffer without the need to correct for solvent shift.

The solubility of the compounds in the assay buffer is an important consideration for small molecule screening studies. Even in the presence of 10% DMSO, many compounds precipitate from the buffer. Such compounds can easily foul up the fluidic channels of flow systems such as Biacore and require extensive cleaning or even loss of the chip. Additionally, such a foul-up will affect the binding of all downstream compounds in the screen. Octet systems use a dip and read method wherein the biosensors dip into sample in the microwell plates.  Only compounds that bind to or dissociate from the biosensor generate a response profile.  The system is largely unaffected by other events in the surrounding medium. This method allows the Octet system to successfully bind compounds from precipitating solutions without fouling the biosensors.

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