Foot-and-mouth disease virus (FMDV) vaccines induce antibodies that protect animals from infection. This study uses bio-layer interferometry (BLI) to characterize the strength of interactions between FMDV antibodies and antigenic sites on the virus particle. BLI measures changes in light as antibodies bind to antigen-coated sensors. The study uses synthetic peptides and artificially generated empty virus capsids as antigens to measure affinity and avidity of antibodies against antigenic site 1 (the G-H loop). Preliminary results show BLI can detect binding of monoclonal antibodies to G-H loop peptides. Future work will correlate BLI measurements of affinity and avidity with virus neutralization tests and vaccine protection levels.

1. Exploring foot-and-mouth disease virus antibody affinity
using bio-layer interferometry
Introduction
Foot-and-mouth disease virus (FMDV) vaccines protect animals from infection by inducing antibodies. In general, the level of FMDV-specific antibodies as measured
by a virus neutralisation test positively correlates with the level of protection afforded against subsequent challenge. As well as overall titre, the strength of antibody
binding can influence an antibody’s neutralising potential. Antibody avidity therefore represents a crucial parameter with regards to the protection afforded by an
antisera and is thus a metric that needs quantification. Avidity ELISAs have been used to measure the avidity of serum for selected FMDV serotypes. An alternative,
label-free method commonly used to evaluate the binding affinity of antibodies is Bio-Layer interferometry (BLI). Here we describe the use of BLI to characterise the
strength of interactions between FMDV antibodies and antigenic site 1 on the G-H loop of the FMDV particle.
Measuring antibody affinity
Antibody affinity refers to the strength of the
interaction between an antibody and its target
epitope. Avidity refers to the overall binding
strength afforded by antibodies based upon their
overall binding (Figure 1).
Polyclonal serum contains multiple antibodies,
each targeting part of the FMDV capsid, the sum
of which can be regarded as the avidity. BLI
measures antibody affinity (avidity in the case of
polyclonal serum) by detecting changes in a light
beam as a result of inter-molecular interactions.
A sensor is loaded with the antigen (ligand),
subjected to a baseline measurement, and then
dipped in the target antibody (Figure 2).
Site I – the G-H loop
Classical monoclonal escape assays have
consistently revealed the presence of five key
neutralizing antigenic sites on the surface of the
FMDV capsid. Site I is a trypsin sensitive loop
containing an Arg-Gly-Asp (RGD) motif
responsible for interacting with the integrin
receptor on host cells.
As it is a linear epitope, an in vitro synthesized
peptide can be used as a surrogate to measure
interactions against this site specifically. Here, we
used a commercially synthesised, biotinylated
peptide as the antigen in BLI experiments. The
peptide was first loaded onto streptavidin coated
Octet sensors – the equivalent of coating an
ELISA plate.
A whole capsid approach
The G-H loop peptide represents a convenient
and useful ligand for use in BLI experiments.
Specifically, it will enable the avidity against this
crucial antigenic site to be measured. An
alternative approach is to utilise artificially
generated empty capsids to represent the entire
FMDV particle.
O1 Manisa capsids have been successfully
biotinylated and loaded onto Octet sensors
(Figure 7). Accurately measuring the avidity of a
polyclonal serum is complex due to every capsid
having multiple copies of multiple epitopes.
However, off-rates are frequently used in place
of KD for ranking antibodies.
Conclusions and future work
• The G-H loop can successfully generate signal
in BLI experiments using antibodies to FMDV.
• Entire capsids offer a manner by which to
assess overall avidity of a serum against the
FMDV capsid.
• Off rates may offer a suitable metric regarding
the strength of a serum.
• How well do KD values and/or off-rates
correlate with VNT and protection afforded by a
serum?
Figure 1.
Figure 2.
Figure 6.
Octet probe preparation
A type O G-H loop peptide was tested at various
dilutions in order to obtain a good level of loading
onto the Octet sensor such that differences could
be measured. A 2-fold dilution series was
prepared, starting at 600nM, and loaded for 900
seconds. After a baseline, the loaded sensors
were dipped into a solution containing the D9
monoclonal antibody (Figure 3). Binding to the
peptide was observed at every concentration
reflecting the sensitivity of BLI.
Modelling of the response by the Octet was shown
to reflect the binding signal measured during the
experiment. Signals were normalised to the
beginning of the association step.
Determining binding to a
monoclonal antibody
An optimal level of loading was used in further
experiments to investigate binding to the D9
monoclonal antibody. D9 is a neutralising
monoclonal antibody whose epitope has been
mapped to the G-H loop. A dilution series of D9
(100, 50, 25 and 12.5nM) was tested using Octet
sensors loaded with 10nM peptide resulting in a
series of curves with which to calculate affinity.
Figure 4.
Figure 5.
Figure 3.
Acknowledgements
We thank Dr Emiliana Brocchi and colleagues
(IZSLER) for the supply of monoclonal antibodies
for use in these experiments.
Funding was provided by Defra and the BBSRC.
Loading phase
100
50
25
12.5
nM
association dissociation
Figure 7.