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A P P L I C A T I O N N O T E 1
Characterization of monoclonalantibody epitope specificity using
Biacore’s SPR technology
Epitope mapping using monoclonal antibodies (MAbs) is a powerful tool in
examining the surface topography of macromolecules. Through its binding, each
MAb defines one specific site, or epitope, on the antigen, and a pair of MAbs which
bind to closely situated epitopes will interfere sterically with each other’s binding
[1,2,3]. Determination of epitope specificity is an important part of MAb
characterization for both investigative work and medical and industrial
applications.
The epitope specificity of a panel of MAbs is most easily determined by testing the
ability of pairs of MAbs to bind simultaneously to the antigen. MAbs directed
against separate epitopes will bind independently of each other, whereas MAbs
directed against closely related epitopes will interfere with each other’s binding.
The most common technique for determining epitope specificities tests pair-wise
binding with RIA or ELISA [4]. One antibody is attached to a solid substrate, the
antigen is bound, and the ability of the second antibody to bind to the surface-
attached complex is tested. A drawback with these methods is that the secondary
interactant must be labelled in some way. Simultaneous binding, indicating distinct
epitopes, is readily identified, but it is generally more difficult to interpret an
absence of simultaneous binding.
This Application Note describes the characterization of epitope specificity patterns
of 30 different MAbs directed against recombinant HIV-1 core protein p24.
Biacore’s SPR technology [5,6] based on surface plasmon resonance (SPR) [7,8] is
used to measure binding of macromolecular components to each other at a sensor
chip surface.
Biacore’s SPR technology based on surface plasmon resonance technology has been
used to map the epitope specificity patterns of 30 monoclonal antibodies against
recombinant HIV-1 core protein p24. The technique does not require labelling of
either antibodies or antigen, and all specificity determinations were performed with
antibodies in unfractionated hybridoma culture supernatants. Pair-wise binding tests
divided the 30 antibodies into 17 groups, representing 17 epitopes on the antigen.
Abstract
Introduction
The principle of the specificity deter-
mination is the same as that described
for RIA- or ELISA-based techniques, but
the use of SPR offers several important
advantages:
• None of the interacting components
needs to be purified or labelled in any
way. As a result, the mapping can be
performed using small amounts of
unfractionated MAbs in cell culture
supernatants.
• A mass-dependent SPR response is
obtained from the binding of each
component to the sensor surface [9].
All stages in the binding process can
thus be monitored.
• Each stage of the binding sequence is
easily quantified, aiding the interpret-
ation of the results.
• The technique allows multi-site
specificity tests using a sequence of
several MAbs.
• The average assay time is short (15
minutes), and large numbers of analyses
can be processed automatically.
Materials
SPR measurements were performed using a
Biacore® system. Sensor Chip CM5 and
Amine Coupling Kit for immobilization
were from Biacore AB.
Immunosorbent purified rabbit anti-mouse
IgG1 (RAMG1), hybridoma culture
supernatants containing murine MAbs
against recombinant HIV-1 p24, and
monoclonal anti-human alpha-fetoprotein
(a-AFP) were obtained from Pharmacia
Diagnostics AB, Uppsala. Recombinant
HIV-1 core protein p24 was supplied by
Pharmacia Genetic Engineering Inc., San
Diego.
SPR response is measured in resonance
units (RU). For most proteins, 1000 RU
corresponds to a surface concentration of
approximately 1 ng/mm2 [9].
Immobilization of RAMG1 on the
sensor chip
RAMG1 was covalently coupled to a
Sensor Chip CM5 via primary amine
groups using the conditions listed in Table
1. The resulting sensorgram (Figure 1)
shows that RAMG1 corresponding to
about 12000 RU is covalently linked to the
sensor chip surface.
Pair-wise binding of MAbs
Pair-wise binding of MAbs to p24 was
tested using the conditions shown in Table
2. Each analysis cycle concludes with
removal of all non-covalently bound
material from the sensor chip surface,
regenerating the surface in preparation for
a new cycle. One cycle takes approximately
15 minutes to perform and in this example,
60 cycles were run automatically.
Materials and methods
Table 1Procedure for immobilizingRAMG1 on a Sensor ChipCM5, to make a specificsurface for adsorption of MAbsfrom hybridoma supernatants.Buffer flow is maintained at 5 µl/min throughout theimmobilization protocol.
Figure 1Sensorgram obtained fromimmobilization of RAMG1 on a Sensor Chip CM5. Numberson the sensorgram indicateinjections as follows: (1) NHS/EDC, (2) RAMG1, (3) ethanolamine, (4) HCl.
Note that the SPR signal is offscale at the top of the RAMG1peak, while the RAMG1solution is in contact with thesensor chip.
Reagents
HBS-EP buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mMEDTA, 0.005% Surfactant P20
NHS: 100 mM N-hydroxysuccinimide in H2O
EDC: 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in H2O
RAMG1: RAMG1, 30 µg/ml in 10 mM Na-acetate pH 5.0
Ethanolamine: 1 M ethanolamine hydrochloride, adjusted to pH 8.5with NaOH
HCl: 100 mM HCl
Biacore immobilization protocol
0 min HBS-EP, flow 5 µl/min Start cycle
5 min Mix NHS + EDC 1:1 Activate surfaceInject 30 µl
11 min Inject 30 µl RAMG1 Couple RAMG1
19 min Inject 30 µl ethanolamine Deactivate excessreactive groups
26 min Inject 15 µl HCl Remove non-covalently bound material
30 min –– End cycle
Table 2Procedure for testingsimultaneous binding oftwo MAbs to p24. Buffer flowis maintained at 5 µl/minthroughout the analysisprotocol.
Figure 2Example of a sensorgramobtained from epitopespecificity determination fortwo MAbs directed againstindependent epitopes. TheSPR response gives theamount of surface-boundcomponent at each stage asfollows: (A) baseline signal,(B)-(A) first MAb, (C)-(B)blocking antibody, (D)-(C) p24,(E)-(D) second MAb.
Reagents
HBS-EP buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mMEDTA, 0.005% Surfactant P20
Salt-free HBS: HBS with NaCl omitted
First MAb: Undiluted hybridoma supernatant containing firstMAb
Blocking Ab: α-AFP, 50 µg/ml in salt-free HBS p24: p24, 10 µg/mlin 10 mM Na-acetate, pH 5.0
Second MAb: Undiluted hybridoma supernatant containing secondMAb
HCl: 100 mM HCl
Biacore analysis protocol
0 min HBS-EP, flow 5 µl/min Start cycle
1 min Inject 4 µl first MAb Bind to RAMG1
4 min Inject 4 µl blocking Ab Block free RAMG1 sites
7 min Inject 4 µl p24 Bind antigen to first MAb
9.5 min Inject 4 µl second MAb Test binding
13 min Inject 10 µl HCl Regenerate surface
15 min –– End cycle
Figure 2 shows a typical sensorgram from
pair-wise epitope specificity studies. The
MAbs tested show simultaneous binding,
and are therefore judged to bind to
independent epitopes.
It is essential that unoccupied RAMG1
sites on the sensor chip surface are blocked
before injection of the second MAb super-
natant, to avoid false positive responses.
This is assured by using a concentration of
blocking antibody sufficient to saturate the
surface even in the absence of the first
MAb. Although different first MAbs
bound to different extents, the SPR signal
level reached after injection of the blocking
MAb was the same regardless of the
amount of first MAb bound. This confirms
that the first MAb and blocking antibody
together occupy all the available sites.
Two kinds of control experiment ensure
that the second MAb binds to the antigen
and not to the RAMG1 or another
component on the sensor chip surface:
• Omission of p24 from the normal assay
sequence reduces the response from the
second MAb supernatant to background
levels. For each supernatant, the mean
background obtained with four
arbitrarily chosen first MAbs was
subtracted from all responses (typical
background levels are 30-100 RU).
• Binding of both purified MAb and p24
is eliminated if blocking antibody is
injected before the first MAb. This also
shows that exchange between surface-
bound blocking antibody and MAb in
free solution is negligible on the time
scale of one assay cycle.
In all, the epitope specificity of 30 different
MAbs was characterized. Theoretically,
this requires 900 tests for the complete
map if all pairs are to be tested in both
binding sequences. In practice, however,
many of the pairs will be redundant, since
a positive result in the first sequence tested
indicates distinct epitopes. Reciprocal pair
Results
tests were run only when a negative result
was obtained, to ensure that the absence of
binding was not an artefact of the sequence
of attachment. The final complete mapping
analyzed 537 binding tests, of which 185
were reciprocal duplicates with the same
antibodies in reversed order.
Four of the MAbs gave negative results
when used as the first antibody, regardless
of which MAb was tested as the second
antibody. Closer examination of the
sensorgrams showed that these MAbs lost
the ability to bind antigen when they were
attached to the surface through RAMG1,
although positive binding was seen in
many cases when these MAbs were used as
second antibody.
These observations illustrate two
particularly valuable features of Biacore’s
SPR technology in comparison with other
epitope mapping techniques: the reason for
the negative response (lack of antigen
binding) is directly apparent from the
sensorgram, and reversed-order pair-wise
tests are easily performed.
The reactivity patterns for the MAbs tested
are shown as a 30x30 matrix in Figure 3.
Figure 3Reactivity pattern matrixshowing the bindingability of pairs of MAbs to p24.
Grouping MAbs that show the same
reactivity pattern gives 17 groups
representing epitopes (Figure 4), which
may be visualized in a two-dimensional
‘‘surface-like’’ map shown in Figure 5.
Note that the diagram does not necessarily
correspond to a physical map of the
binding sites on the antigen surface, since
conformational changes in the antigen or
electrostatic interactions between MAbs
may distort the binding patterns. In this
particular case, however, the results do not
contradict a simple two-dimensional
‘‘surface-like’’ interpretation of the map.
Biacore’s SPR technology can easily be applied
to multi-determinant binding experiments, in
addition to the simpler pair-wise binding
tests. An example of a sequential multi-
determinant test is shown in Figure 6.
Here, with p24 linked to the surface
through MAb 31, MAbs 41 and 44 are
both prevented from binding, while MAbs
17, 33, 23, 5 all bind independently of
each other in that order. The last antibody,
MAb 7, does not bind, as expected from
the pair-wise exclusion of MAbs 5 and 7.
These results accord well with the
conclusions from the epitope specificity
studies. Note that in this type of
experiment, saturation of the surface
binding sites at each stage is essential. Each
MAb was therefore injected over a longer
time period than for the pair-wise binding
tests, until a plateau was reached in the
SPR signal.
Figure 4Grouping 30 MAbsaccording to theirreactivity patternsidentifies 17 proposedepitope regions.
Figure 5Two-dimensional‘‘surface-like’’ map ofthe epitopes based onthe matrix in Figure 4.Overlapping circlesrepresent MAb groupswithin which pairs ofMAbs cannot bindsimultaneously.
Figure 6Multi-determinantbinding of MAbs top24. The MAbs injected at each stageare identified withreference to thediagram obtained fromtwo-site specificitystudies.
The work in this Application Note
demonstrates that Biacore’s SPR
technology can be used to characterize
epitope specificity with MAbs in
unfractionated hybridoma culture
supernatant. The quantitative data
obtained for each step in the binding
process permits a more comprehensive
interpretation of the binding than is
possible with conventional techniques.
Although this study concerned only levels
of antibody binding, the progress of each
binding step in real time is automatically
recorded, so that both kinetic and
equilibrium parameters may be assessed
for macromolecular interactions. The
technique is well suited to programmed
operation, and can handle many samples
without user intervention. This feature is
important in epitope specificity
determination of a large panel of MAbs,
where the pair-wise combination matrix
requires a large number of assay cycles.
Results
References
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2. Krummenacher, C. et al. J Virology 74; 10863(2000)
3. Novotny, L. A. et al. Infect Immun 68; 2119(2000)
4. Goding, J.W., Monoclonal Antibodies:Principles and Practice (Academic Press, London,1983).
5. Fägerstam, L.G., Techniques in ProteinChemistry II, ed. J. J. Villafranca, pp. 65-71(Academic Press, New York 1991).
6. Jönsson U. et al., BioTechniques 11; 620(1991).
7. Kretschmann, E. and Raether, H., Z.Naturforschung, Teil. A 23; 2135 (1968).
8. Liedberg, B., Nylander, C. and Lundström, I.,Sensors and Actuators 4; 299 (1983).
9. Stenberg, E. et al., J. Colloid and InterfaceScience 143; 513 (1991).
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