Best Practice Guide

April 14, 2022
Keywords or phrases:
assay design, buffer optimization, electrostatic, nonelectrostatic, non-specific binding

Best Practice Guide:
Minimizing the Effectsof Non-specific Binding
Stuart Knowling Ph.D. Senior Scientist
Sartorius Corporation, 47661 Fremont Blvd, Fremont, CA 94538 USA
Correspondence
Email: octet@sartorius.com

600500Non-specific binding (NSB) of biological and non-biologicalmolecules to the sensor chip surface in surface plasmon
resonance (SPR) assays can be problematic and cause errors
in determining accurate kinetics and affinity. Non-specificbinding is characterized by a significant binding to thereference surface of the sensor chip, which does not return
to baseline at the end of injection (Fig 1).
In theory, non-specific binding can be ‘referenced’ out if theactive and reference surfaces exhibit very similar levels of
non-specific binding but in practice, this is not feasible asligand immobilization may mask non-specific binding on theactive channel, and subsequently lead to higher levels ofobserved non-specific binding on the reference surface.

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Response (RU)

Introduction

400300200100

Reference Surface

00100

200300400
Time (seconds)

500600Figure 1
Example of Non-specific Binding
Reference surface (pink) exhibits significant curvature during the analyte
injection and does not return to zero at the end of the injection, indicating
non-specific binding.

Electrostatic and Non-electrostatic
Non-specific Binding

Assay
DesignTwo common types of non-specific binding are observedin SPR assays:
Electrostatic NSB
Non-electrostatic NSB

To collect accurate data, non-specific binding must be
considered and tested during assay design to determine
whether non-specific binding is occurring and if yes, what
measures can be used to remove the observed nonspecific binding.

Electrostatic NSB is characterized by attraction of thepositively charged analyte to the negatively charged
sensor chip surface (Fig 2). This is often exacerbated whenusing carboxymethyl dextran-based sensor chips due totheir net-negative charge. Attraction of the positively
charged analyte to the sensor chip surface often occurs
when the analyte has a higher isoelectric point than thatthe running buffer.

Ideally a “square-shaped sensorgram”, reflecting any slightmismatch in bulk refractive index between sample andrunning buffer should be observed during the association
phase (Fig 4).

Analyte(+)

Analyte(+)

Analyte(+)
LigandAnalyte(+)

Ligand(-) (-) (-) (-)

600
Ligand(-) (-) (-) (-)

Figure 2
Electrostatic Binding
Electrostatic NSB is characterized by attraction of the positively charged
analyte to the negatively charged sensor chip surface.

Non-electrostatic NSB is characterized by interaction of
the analyte or components of the sample to the sensor
chip through such interactions as hydrophobic, hydrogenbonding or biding to exposed gold patches on the sensor
chip (Fig 3).
Non-electrostatic NSB is particularly noticeable withthiol-containing small molecules and ‘sticky’ or crude
samples that contain patches of hydrophobicity or charge
on the protein’s surface.
Hydrophobic Patch
AnalyteAnalyteAnalyte
LigandLigandLigandAnalyteFigure 3
Non-Electrostatic Binding
Analyte containing large hydrophobic patches (black region) aresusceptible to non-electrostatic NSB.

LigandResponse (RU)

Ligand2In general, the simplest way to determine a molecules
proclivity for non-specific binding is to inject the highest
concentration of the analyte across an unmodified sensor
chip surface. It is critical that the same sensor chip that isplanned to be used during the assay is tested to ensure an
accurate result.

400200050

100150200Time (seconds)

Figure 4
Ideal Sensorgram Shape
Interaction of 20 µM of a small molecule with carbonic anhydrase II
shows no observable NSB and a square-shaped sensorgram.

If the sensorgram shows any “tailing” during thedissociation phase, then the analyte may be interacting
non-specifically with the surface and strategies forlowering the analyte’s non-specific binding with thesensor chip surface must be used.
In general, strategies for dealing with non-specific binding
can be separated into two basic strategies:
Sensor chip optimization
Buffer optimization

Sensor Chip Optimization
As discussed previously, the net negative charge ofcarboxymethyl dextran-based sensor chips can cause
electrostatic non-specific binding to be observed andtherefore, it is recommended that different sensor chiptypes with fewer functional groups are tested for a
reduction in NSB. For example, if an Octet® SPR CDH
sensor chip produces an observable level of non-specificbinding then an Octet® SPR CDL sensor chip, whichcontains fewer carboxylic groups should be tested and/ora planar sensor chip such as the Octet® SPR COOH1
sensor chip, which contains no carboxymethyl dextran
should be considered (though the user must consider therequired immobilization levels when assessing alternativesensor chips).
As discussed in the best practice guide “Minimize Artifacts”,
correct reference surfaces have a large impact on dataquality in addition to having a noticeable effect on nonspecific binding. Where possible the reference surface
should contain the same immobilization conditions as theactive surface and contain a non-interacting protein thatshould be immobilized to the same response level as theactive surface. The non-interacting protein does notdirectly reduce non-specific binding but masks the charge
on the sensor chip surface and aids in allowing non-specificbinding to be ‘referenced’ out.
Where a positively charged analyte is being assessed it ispossible to lower the sensor chips net negative chargeduring amine coupling-based immobilizations by using thesame assay parameters and inactivating remaining
succinimide esters with ethylenediamine instead of
ethanolamine. This reduces the net negative charge on thesensor chip surface and decreases the potential for nonspecific binding.

Buffer Optimization

Where non-specific binding is still observed it is thenpossible to change the buffer choice or add components tothe running buffer to reduce non-specific binding. The
addition of 0.5 – 2.0 mg/mL (0.05 - 0.2 %) BSA to therunning buffer can block potential non-specific bindingsites and helps stop proteins stick to assay plates andtubing.
The addition of NaCl up to a final concentration of 500
mM in the running buffer can reduce electrostatic nonspecific binding, though care must be taken that theanalyte or ligand activity is not affected in nonphysiological salt levels.
When dealing with ‘sticky’ or crude samples it isrecommended that the sample is diluted into the runningbuffer and NaCl levels are investigated. Although a dilution
factor of five is a common recommendation, it is advisedthat the user determines the optimum dilution level andprotocol through empirical means.
Where possible the analyte should be as pure as possible toensure that contaminants that may cause non-specificbinding are removed. Where it is not possible to eliminate
non-specific binding using the techniques shown here,orientating the assay with the ‘sticky’ protein as the ligand
should be considered. Attention must be paid to theimmobilization process where this is attempted as theactivity of the immobilized ligand may be affected by its
non-specific binding.

Conclusion
Minimizing non-specific binding is often not factored into
assay development despite it being a significant factor in
the generation of high-quality, accurate data. Therefore, it isrecommended that non-specific binding is assessed inassay development and that the best practices outlined inthis document are implemented where non-specificbinding is observed.

The choice of running buffer is of critical importance in SPR
assays as it is the carrier of the analyte and therefore, anycomposition, pH or other excipient issues can affect theinteraction between the analyte and ligand. Assessment of
non-specific binding should be performed in a suitablebuffer such as HBS-EP+.
In general, it’s recommended to start with the ‘plus’ versionof the running buffer as the additional tween (~0.05%)
prevents non-specific sticking to the systems micro-fluidics
and tubing, which helps reduce both electrostatic and nonelectrostatic "stickiness" of samples.
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