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Ideally, the goal of assay development should be to eliminate all binding of non-analyte components, so that the assay is entirely specific for analyte. This is however not always feasible, and the approach to dealing with non-specific binding of this kind depends largely on the situations in which the assay will be used.

Problems with non-specific binding can in general be addressed in three ways: design of the experiment, choice of sensor surface and addition of NSB Reducer (available from Biacore) to the sample.

Design of the experiment

Apart from the obvious approach of partially purifying the sample to remove components that interfere with the assay, non-specific binding may in some cases be reduced by optimizing the composition of the running buffer. In general, physiological (150 mM) or higher salt concentrations will help to suppress non-specific electrostatic interactions, and inclusion of salt in the running buffer is always recommended. Other buffer components may have a significant effect on non-specific binding in individual cases.

A powerful technique for dealing with non-specific binding in applications that measure binding levels (as opposed to kinetic interaction profiles) is to use enhancement reagents to specifically amplify the signal from the analyte. An enhancement reagent binds to the analyte independently of the ligand, so that the enhancement response is a direct indication of the amount of analyte on the surface. In this way the analyte is detected and identified with a double specificity, once by interaction with the immobilized ligand and once by interaction with the injected enhancement reagent.

Choice of sensor surface

Different sensor chip types have different characteristics with respect to non-specific binding. It is difficult to provide general recommendations in this respect, since the effects vary according to the nature of the sample: for example, Sensor Chip CM4 tends to show lower non-specific binding than Sensor Chip CM5 with cell culture medium and crude cell extracts, but the reverse is often true with serum and plasma. As a general guideline, if non-specific binding is a problem, it can be worth testing the application on a different sensor chip type.

Similarly, the ligand immobilization chemistry can influence the level of non-specific binding in an unpredictable manner. When the ligand is amenable to alternative immobilization chemistries, testing a range of different immobilization methods can help to identify conditions that reduce non-specific binding.

In some applications, immobilization of aminomethyl-polyethyleneglycol (aminomethyl-PEG) to the surface prior to ligand immobilization has been found to reduce the levels of non-specific binding. To use this approach, activate the surface with a 10-minute injection of 0.05 M EDC/0.2 M NHS. Follow this activation with an injection of 5-10 mM aminomethyl-PEG in 10 mM sodium borate pH 8.5 for 5–10 minutes. Immobilize the ligand using the chosen chemistry directly after the aminomethyl-PEG injection.

The immobilization capacity of the surface for ligand is reduced after immobilization of aminomethyl-PEG, and a balance has to be struck between the level of aminomethyl-PEG substitution and the remaining capacity of the surface. Acceptable results have been observed with levels of aminomethyl-PEG around 500-2000 RU.

Where alternative ligands are available (for example with monoclonal antibody ligands), the choice of ligand may have a decisive effect on the extent of non-specific binding. In many cases, immobilization of Fab'2 fragments can be preferable to the use of intact antibodies, eliminating binding of sample components to the Fc portion of the antibody.

Sample additives

NSB Reducer (available from Biacore) added to the sample at 0.5–1 mg/ml can compete for molecules that bind to the dextran on the sensor surface without interfering with the analyte-ligand interaction.

In some cases, addition of “ligand mimics” can help to reduce unwanted binding to the immobilized ligand. An example of this approach is the addition of polyclonal antibody preparations (at 100–200 µg/ml) from non-immune animals to counteract non-specific binding to immobilized monoclonal antibodies.