Best Practice Guide

April 14, 2022
Keywords or phrases:
Amine Coupling, Affinity Tags, Avidity, Stress Molecules,
Structure Function RelationshipBest Practice Guide:
Octet SF3 Assay Orientation®

Stuart Knowling Ph.D. Senior Scientist
Sartorius Corporation, 47661 Fremont Blvd, Fremont, CA 94538 USA
Correspondence
Email: Octet@Sartorius.com

Introduction
In surface plasmon resonance (SPR), irrespective of the chemistry used (coupled or captured) one
binding partner is injected in solution (as analyte) over a specific binding partner, which is immobilized
or captured on the sensor chip (as ligand).
In general, assay orientation plays an important role in SPR assays and should be designed with thesimplest biologically relevant model in mind. Oftentimes the inability to fit data to a simple model canbe a result of assay design and not a systemic flaw in the technology.

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The Impact of Different Immobilization
and Capture Approaches
Although there are many types of chemistry available forcovalently attaching proteins to SPR sensor chips, themost commonly used is amine coupling. Benefits of aminecoupling include its stability and that the protein does nothave to be modified in any way. In addition, the amount ofligand required is low and it is generally easy to achieve thesame immobilization level in subsequent assays.
Many proteins have accessible primary amines on theirsurface, which means that numerous orientations of theprotein can be covalently attached to the sensor chipsurface. Therefore, the main drawback associated withamine coupling is the creation of a heterogenous sensor
chip surface. As shown in the Figure 1, this can cause
obstruction or blocking of binding sites. Additionally, low
pH solutions that are required to drive the protein towards
the chip surface during amine coupling can cause protein
degradation. Another drawback associated with amine
coupling is the need for high purity ligands as impurities, if
amine bearing, can co-immobilise to the sensor chipsurface and reduce the binding capacity.

Figure 1
Amine Coupling

reagents, affinity tags allow lower purity preparations to beused. During affinity capture, only those molecules bearing
the affinity tag will be captured while impurities remain in
the bulk flow and are removed under standard runningbuffer flow.

His-Tag
His-Tag, containing 6 or more Histidine residues at the Nor C-terminal, is one of the simplest and most frequently
used in affinity purification and capture assays.
The most common way to capture a His-Tag ligand isusing an anti-histidine antibody, which can detect mixed
His-Tags (non-selective), which allows the use of one
antibody for all assays, or antibodies that are selective
against N- or C-terminal His-Tags. The antibody caneasily be regenerated using a low pH Glycine solution
and enables multiple ligand and analyte combinations
to be tested.

Figure 2

LigandLigandNote. Inclusion of a specific affinity tag (magenta dot) such as His-Tag,
AviTagTM or Fc Tag allows oriented capture of the ligand.

Note. Primary amines (black dots) on the ligand can be covalently
attached to carboxymethyl groups present on the sensor chipsurface in multiple orientations, which can hinder or block thebinding site (yellow dot).

It is important to consider that chemically biotinylated
proteins can be captured in a similar ‘random’ orientation
if they contain multiple primary amines. However even if
the protein contains only one primary amine suitable forlabeling then there is the possibility that capture via thebiotin may occlude the binding site.
The most common solution to problems with amine
coupling is to introduce an affinity tag into the ligand whichallows a specific ligand orientation to be created, where allmolecules are orientated in the same way, which helps
prevent any differences in binding site presentation. Inaddition, unlike amine coupling which requires high purity
2

Another common way of capturing His-tagged molecules
is by using a specialized sensor chip such as the Octet®
SPR HisCap sensor chip (Catalog No. 19-0058). Based on
the principle of Immobilized Metal Affinity Chromato­graphy (IMAC), the His-tagged molecule is captured via
Ni2+/NTA chelation and regenerated using EDTA to strip
the metal ions and the captured his-tagged protein away.
The regenerated HisCap chip surface can then be re-usedby recharging the NTA surface with fresh Ni2+.
The main benefit of His-Tag capture is that the surface canbe easily regenerated and, in general, can handle multiple
regenerations before any decrease in capture efficiency orbaseline drift is noted.

Therefore, site specific biotinylation of ligands is highly
desirable to not only create a homogenous sensor chipsurface but also a highly stable ligand surface and baseline.
Many proteins are commercially available in large quantities
and contain a site specific biotin molecule due to theincrease in use of AviTagTM technology, which allows SPR
users to create a homogenous sensor chip surface with an
exceptionally stable baseline on streptavidin preparedsensor chips such as the Octet® SPR SADH sensor chip
(Catalog No. 19-0130).

Orient Assays to Avoid Avidity
Creation of a homogenous ligand bound sensor chipsurface is desirable but other issues such as avidity must be
considered when designing SPR assays. The conventional
bivalent IgG homodimer antibody presents two
independent and equal binding sites for a specific antigen,and therefore, is susceptible to avidity effects if assay
orientation is not considered.
As shown in Figure 3A, when the antibody is injected as theanalyte, each binding site has the potential to bind to theligand and cause avidity issues.

Figure 3A.

B.
AnalyteLigandBiotin-Streptavidin
Biotin is a small molecule that has a very high affinity for
streptavidin (Kd ~10-14 M), which can generate stablebaselines for SPR assays. Labeling of molecules with biotinrarely interferes with function, making it a good choice for
capture, although as discussed in the previous section, thesurface presentation can be heterogenous.

AnalyteLigandUnlike His-Tag capture, regeneration can become anissue and careful consideration must be given to choosea suitable regeneration solution.
LigandFc Tag
IgG-Fc tags have been shown to improve the stability ofproteins and increase the solubility of the yield and are
therefore, a popular choice of affinity tag for commercial
proteins.
The use of Fc Tag as a default tag presents challenges for
SPR assays as, although the Fc tag allows assay-specificorientation through capturing via the Fc tag, measurementof Fc containing proteins, such as IgG antibodies, becomeschallenging, as they also contain the structural motif thatbinds to the Fc-tagged capture molecule. Inversion of theassay, where the antibody is captured using a capture
molecule such as protein A or G also causes the same issue,
as the analyte with an Fc tag will bind to the capture
molecule causing a large amount of undesirable binding.
Standard techniques to reduce non-specific binding
cannot be used as there is a specific capture molecule
there for the Fc region, which will also affect ligand capture.
Therefore, careful consideration must be given to the assay
setup when proteins containing Fc Tags are required for
creating a homogenous sensor chip surface.

LigandLigandNote. Assay orientation choice plays an important part in determining
accurate kinetics and affinity.

Therefore, it is usually advisable to inject the antigen overthe immobilized antibody (Figure 3B) to avoid avidity
artifacts, provided that the antigen is monovalent.
This assay format offers the advantages of both keeping
the antigen in solution to enable access to all epitopes,and scalability, with the Octet® SF3 allowing a large numberof ligands and antigens to be analyzed per assay array,
allowing hundreds of interactions to be analyzed in parallel.

Structure Function Relationships
Samples stored under stress conditions such as, heat, acid/base hydrolysis and oxidation are often used in structure
function relationship studies. Assay orientation canbecome especially important when looking at stressed
samples, as assay orientation may cause the assay to beinsensitive to stress or function alterations due topreferential binding of the non-stressed molecules
over the weaker binding stressed molecules.

3For example, in Figure 4A there is a heterogenous
population of stressed molecules (stress is shown inyellow) as may be found in a short-term stress study. Ifthe antibody is the analyte and the antigen is the ligand
then the un-stressed antibodies will preferentially bindover the stressed molecules (Figure 4B); therefore, theassay is insensitive to measuring the effect on stressedmolecules and may lead to an inaccurate conclusionfrom the data.

Figure 4A.

Calculating Surface Activity
It is desirable to determine the activity of the capturedor immobilized ligand (and as such, its conformationalstructure) during assay development. There is a direct
1:1 relationship between the amount of ligand and theobserved Rmax in SPR assays. For a captured antibody(Figure 6) this is due to an increased number of antigen
binding sites as more mAb is immobilized and as suchcan bind more analyte, which leads to an increase in theobserved Rmax. (Rmax vs Immobilization Level).

B.
Figure 6
Ligands
Ligands

AnalyteAnalyteLigandLigandNote. Unstressed molecules (all black) preferentially bind to the target
when compared to stressed molecules (yellow),which may leadtoLigand
LigandLigandincorrect conclusions being reached.

Ina modified assay setup with the target asAnalyte
the analyte
Analytes
Analytes(Figure 5A), the assay sensitivity to stress or function
alterations is improved due to the non-biased present­ationof the molecule of interest. In this orientation the sensor
chip surface heterogeneity represents the stressedmolecule population more accurately and a decreased
response would be observed compared toonly unstressed
Ligandsmolecule being captured. This assay orientation also offers
Ligands
the advantage of decreasing
avidity effects as describedabove.

Figure 5A.

Analytes

B.

AnalyteNote. Capture-based assays may offer a truer representation ofstress effects.

Analytes

Note. Captured antibodies present two independentand equal binding sites for a specific antigen.

To calculate the surface activity a range of immobilizationlevels must be assessed, and the mass of the ligand andanalyte known. As an example, in Figure 6, the mass of themAb is 75 kDa per antigen binding site and the mass ofmonomeric analyte is 30 kDa.
In an assay setup where 100% of the surface binding wasavailable to the analyte a molar ratio of 0.40 (30 kDa/75kDa) would be observed. Comparing the gradient of theRmax vs Immobilization Level to the predicted molar
ratio allows the user to determine the surface activity.
For example, if an Rmax vs Immobilization Level gradientof 0.2 was determined then a surface activity of 50% isobserved (0.2/0.4*100).
As discussed above, it must be remembered thatstandard immobilization techniques such as amine
coupling create a heterogenous surface and therefore,
will affect surface activity.

Conclusion
Consideration of orientation during assay design canhelp create homogenous surfaces that minimize avidity
artifacts and allow data to be biologically relevant andfitted to the simplest model possible.
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Specifications subject to change without notice.
Copyright Sartorius Lab Instruments GmbH & Co. KG.
For Research Use Only. 4071 RevA
Status 03 | 2022

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