JOURNAL OF MOLECULAR RECOGNITION, VOL.

11, 163–167 (1998)

Measurement of antigen–antibody interactions

with biosensors
Marc H. V. Van Regenmortel,* Danièle Altschuh, Jean Chatellier,† Lars Christensen,‡
Nathalie Rauffer-Bruyère,§ Pascale Richalet-Secordel,} Jean Witz and Gabrielle Zeder-Lutz
IBMC, 15 Rue René Descartes, F-67084 Strasbourg Cedex, France

The introduction in 1990 of a new biosensor technology based on surface plasmon resonance has revolu-
tionized the measurement of antigen–antibody binding interactions. In this technique, one of the interacting
partners is immobilized on a sensor chip and the binding of the other is followed by the increase in refractive
index caused by the mass of bound species. The following immunochemical applications of this new
technology will be described: (1) functional mapping of epitopes and paratopes by mutagenesis; (2) analysis
of the thermodynamic parameters of the interaction; (3) measurement of the concentration of biogically
active molecules; (4) selection of diagnostic probes. # 1998 John Wiley & Sons, Ltd.

J. Mol. Recogn. 11, 163–167, 1998

Keywords: biosensors; BIAcore; epitope mapping; thermodynamics; concentration determination; diagnostic reagents

Introduction been devoted to the many applications of SPR technology in

The introduction in 1990 of a new biosensor technology
based on surface plasmon resonance (SPR) has greatly
simplified the measurement of antigen–antibody binding
interactions. This new technology, known as biomolecular
interaction analysis (BIA), makes it possible to visualize the
binding process as a function of time by following the
increase in refractive index that occurs when one of the
interacting partners, known as the analyte, binds to a ligand
immobilized on the surface of the sensor chip. There is no
need to label the reactants, which avoids the artefactual
changes in binding properties that often result when the
molecules are labelled. In the past, kinetic analysis of
interactions was feasible only with a few systems possessing
unique spectral properties. The availability of biosensors
now makes it possible to investigate the kinetics of antigen–
antibody interactions in a fairly routine manner and our
understanding of the complex nature of these reactions has
increased dramatically as a result (Morton et al., 1995;
Karlsson and Roos, 1997; Saunal et al., 1997). Several
reviews describing this new technology have been published
(Chaiken et al., 1992; Malmqvist, 1993, 1996; Fägerstam
and Karlsson, 1994; Myszka, 1997; Purvis et al., 1997; Van
Regenmortel et al., 1997) and special issues of journals have
* Correspondence to: M. H. V. Van Regenmortel, IBMC, 15 Rue René
Descartes, F-67084 Strasbourg Cedex, France.
† Current address: Centre for Protein Engineering, MRC Centre, Hills Road,
Cambridge CB2 2QH, UK.
‡ Current address: Department of Biochemistry and Nutrition, Technical
University of Denmark, Building 224, DK-2800 Lyngby, Denmark.
§ Current address: Plant Molecular Biology Center, Northern Illinois
University, De Kalb, IL 60115-2861, USA.
} Current address: Genetics Institute, Burtt Road, Andover, MA 01810, USA.
biology and immunology (Granzow, 1994; Van Regenmor-
tel, 1995). In the present review a number of applications of
biosensor measurements in immunochemistry will be
described.
Functional Mapping of Binding Sites
For many years the antigenic determinants or epitopes of
proteins have been described in terms of structural
information obtained by X-ray analysis of antigen–antibody
complexes (Saul and Alzari, 1996). This approach leads to
the definition of so-called structural epitopes comprising
15–20 amino acid residues considered to be in contact with
a complementary antibody-binding site or paratope. Each
paratope is constituted of six complementarity-determining
regions (CDRs) which form loops of hypervariable
sequence located at the end of the two variable domains
of an immunoglobulin molecule (Day, 1990). Although the
approximately 50 residues that contribute to the CDRs of an
immunoglobulin may be viewed as a potential binding
pocket, the structural paratope is made up of only about one-
third of these residues. Each immunoglobulin molecule is
thus clearly multispecific, i.e. able to bind to many
structurally related or unrelated antigens (Richards et al.,
1975). It is actually misleading to describe epitopes and
paratopes as comprising as many as 15–20 residues, since
the interactions occur at the atomic level and it is unlikely
that all or even most of the atoms of a given residue
participate in the interaction. Furthermore, binding mea-
surements with analogues presenting single substitutions of
whole residues (Jin and Wells, 1996) or of atomic groups
(Rauffer et al., 1994) have suggested that atoms from three
to five residues in each of the partners contribute the major

# 1998 John Wiley & Sons, Ltd. CCC 0952–3499/98/010163–05 $17.50

164 M. H. V. VAN REGENMORTEL ET AL.
portion of the binding energy of interaction. In particular,
alanine-scanning mutagenesis is able to provide a systema-
tic analysis of the energetic importance of individual side-
chains (Wells, 1991). This type of functional analysis leads
to the definition of so-called energetic or functional epitopes
that are considerably smaller than structural epitopes
(Benjamin and Perdue, 1996).
A number of recent studies of the binding properties of
recombinant Fab fragments using biosensor technology
have demonstrated that this technique is able to detect very
small differences in binding kinetics (Chatellier et al.,
1996a). In one study the influence on binding of 13
conserved residues at the interface between the VL and VH
domains of a monoclonal antibody (Mab 57P) specific for a
viral octapeptide was analysed by alanine scanning
(Chatellier et al., 1996b). It was found that the replacement
of bulky side-chains had the most drastic effect on the
kinetic rate constants of the peptide–Fab mutant interac-
tions, but that the replacement of smaller side-chains also
had a significant effect. Modification of some of these
residues which are located 9–12 Å away from the base of the
CDR loops is unlikely to have altered loop conformation, so
the observed differences in binding were probably due to a
change in the relative position of the VL and VH domains.
These results confirm previous data showing that residues
remote from the paratope are able to influence the antigen-
binding properties of antibodies (Lavoie et al., 1992; Kelley
and O’Connell, 1993; Schildbach et al., 1993).
In a subsequent study the same recombinant Fab fragment
57P was used to produce hybrid molecules with another
recombinant Fab (174P) which binds to a very similar
epitope. Kinetic measurements had shown that the Fab 174P
dissociated faster from various peptide variants of the
epitope than Fab 57P (Chatellier et al., 1992a). In order to
identify which of the 15 residues that were different in the
two Fabs were responsible for the different off-rates, six
hybrid Fabs were constructed by exchanging three DNA
segments, In addition, four mutants were obtained by site-
directed mutagenesis (Rauffer-Bruyère et al., 1997). The
high precision of biosensor measurements made it possible
to show that at least five substitutions changed the
dissociation kinetics in a small but significant manner.
The kinetic data also demonstrated the presence of small-
magnitude non-additive effects of multiple substitutions.
These results support the notion that protein residues act
cooperatively on activity (Sturtevant, 1994) and that
residues located away from each other and away from the
paratope are able to influence the dissociation kinetics of
antigen–antibody interactions. The small deviations from
additivity with respect to dissociation kinetics could be
demonstrated unambiguously because of the high reprodu-
cibility of biosensor data which made it possible to reliably
detect 10%–20% differences in dissociation rate constants
(Rauffer-Bruyère et al., 1997).
Thermodynamic Analysis of Binding
Parameters
The thermodynamic parameters of an antigen–antibody
interaction can be determined by measuring the temperature
# 1998 John Wiley & Sons, Ltd.
dependence of its kinetic association and dissociation rate
constants. Following the introduction in 1995 of the
BIAcore 2000 instrument (Karlsson and Ståhlberg, 1995),
it is now possible to measure kinetic rates with high
precision at temperatures ranging from 5 to 40 °C. Changes
in enthalpy (DHo) can be calculated directly from kinetic
constants (Raman et al., 1992) or from the equilibrium
association constant in the standard state (1 M concentra-
tion):

Go ˆ
H o ÿ T
S o ˆ ÿRT lnK o
If the enthalpy change DH does not vary with temperature,
the van’t Hoff plot of lnK vs 1/T results in a straight line of
slope ÿDH/R:
lnK ˆ ÿ
H=RT ‡
S=R
In the case of protein–protein interactions, DH and DS
values often show a significant temperature variation,
resulting in a non-zero value of the heat capacity change
DCp:

Cp ˆ d…
H†=dT ˆ Td…
S†=dT
In order to include the temperature variation of enthalpy in
the van’t Hoff equation, the integrated form of the equation
should be used (Naghibi et al., 1995):
lnK ˆ lnKo ‡ …
Ho ÿ To
Cp =R†…1=To ÿ 1=T†
‡
Cp =R ln…T=To †
In a recent study the interaction between hen egg white
lysozyme (HEL) and the Fab fragment D1.3 was analysed at
temperatures between 5 and 40 °C using biosensor technol-
ogy (Zeder-Lutz et al., 1997). To avoid mass transport
limitations, very small quantities of HEL (30–40 RU) were
immobilized on the sensor chip and sensorgrams were
obtained using between 3 and 120 nM of Fab D1.3 as
analyte.
The ka value for the HEL–Fab D 1.3 interaction at 25 °C
was 1.0  106 Mÿ1 sÿ1, a value very close to that found
earlier using stopped flow fluorescence spectroscopy (Foote
and Winter, 1992). The value of DG at 25 °C (ÿ49 kJ Mÿ1)
was very close to the value of ÿ48.5 kJ Mÿ1 calculated by
Bhat et al. (1994) from fluorescence-quenching data.
However, the value of DH measured at 25 °C by biosensor
technology (ÿ35 kJ Mÿ1) was much smaller than the value
determined by calorimetry (ÿ90 kJ Mÿ1) by Bhat et al.
(1994). Two other differences were observed, i.e. a limited
variation in lnK and DG with temperature determined by
BIAcore compared with the steady decrease in lnK with
temperature found by calorimetry, and a positive value of
DCp (about 4.0 kJ Mÿ1 Kÿ1) compared with the ÿ1.6 kJ
Mÿ1 Kÿ1 determined by microcalorimetry.
Although differences in DH values obtained by calori-
metry and by use of the van’t Hoff equation have been
reported previously (Naghibi et al., 1995), the magnitude of
the differences observed in the HEL–Fab D1.3 system is
surprising. Isothermal calorimetry measures directly the
enthalpy of formation of the complex without making any
assumption on the mechanism of the reaction (Jeserelov et
al., 1996). In contrast, the use of the van’t Hoff equation
implies that the reaction is of a simple bimolecular type,
which may not be valid in this case. Furthermore, the
JOURNAL OF MOLECULAR RECOGNITION, VOL. 11: 163–167 (1998)
 MEASUREMENT OF INTERACTIONS WITH BIOSENSORS
presence of bound water at the interface between HEL and
Fab D1.3 (Bhat et al., 1994; Goldbaum et al., 1996) may
also have influenced the thermodynamic parameters, as was
found in the case of protein–DNA complexes (Morton and
Ladbury, 1996). Others studies with systems having smaller
equilibrium constants than the D1.3 antibody, which would
allow DG to be determined by both calorimetry and
BIAcore over a wide range of temperatures, are needed to
unravel the source of these discrepancies in observed DH
values.
Determination of the Concentration of
Biologically Active Molecules
Most methods for determining protein concentration are
based on chemical procedures that are not applicable to
unpurified samples and suffer from the limitation that they
do not distinguish between biologically active and inactive
molecules. In contrast, the biosensor technology is able to
measure the concentration of biologically active molecules
in a crude preparation. If mass transport of the protein
analyte from the bulk flow to the sensor surface is much
faster than the binding to the immobilized ligand, the
concentration of analyte at the surface will be the same as in
the bulk, and the binding that is observed will exclusively
reflect the binding kinetics. On the other hand, if mass
transport is the only limiting factor, the binding signal will
be proportional to the concentration of active analyte. This
allows the concentration to be determined independently of
the binding kinetics. A method for measuring antibody
concentration based on this approach has been described by
Karlsson et al. (1993). This method requires the immobi-
lization of a large amount of ligand as well as the use of a
calibration curve and cannot be used when the association
rate constant of the interaction is less than 105 Mÿ1 sÿ1.
Recently another more general biosensor method for
measuring protein concentration has been described which
is applicable to conditions where mass transport is not
totally but only partially rate-limiting (Christensen, 1997;
Richalet-Secordel et al., 1997). This method, which is based
on measurements at different flow rates, does not require a
standard of known concentration and is applicable to
systems with an association rate constant as low as 103
Mÿ1 sÿ1.
The concentration of analyte injected at different flow
rates over the sensor surface can be calculated using the
equation
dR=dt ˆ Lm
Lr
MW
G‰Abulk Š=…Lm ‡ Lr†
where R is the response level in resonance units, Lm and Lr
are the Onsager coefficients (Glaser, 1993; Christensen,
1997), MW is the molecular weight of the analyte (g molÿ1),
G is a parameter corresponding to the signal obtained when
1 ng of molecules is captured by the immobilized ligand
over 1 mm2 of the surface (G = 1000 RU mm2 ngÿ1), and
[Abulk] is the analyte concentration (nM).
The dR/dt values can be obtained by linear regression or
by a global non-linear least squares analysis. If dissociation
is not negligible at the beginning of the association, a
correction factor can be introduced (Christensen, 1997). The
# 1998 John Wiley & Sons, Ltd.
165
method requires a minimal binding rate of about 1 RU sÿ1 at
a flow rate of 5 ml minÿ1, while the maximal binding rate
should be about 25 RU sÿ1 at the same flow rate (Richalet-
Secordel et al., 1997).
This simple biosensor method of concentration determi-
nation is likely to find many applications in biotechnology
and biomedical research. It is frequently found that only a
fraction of biological product preparations actually pos-
sesses biological activity and there is a considerable need
for reliable methods to measure the activity of recombinant
proteins, assess the lot-to-lot consistency of batches of
biologicals and control the stability of biomolecules after
transportation or prolonged storage.
Selection of Diagnostic Probes
Synthetic peptides are widely used as diagnostic probes for
detecting viral antibodies produced during infection (Lei-
nikki et al., 1993), and biosensors are a useful tool for
monitoring attempts to optimize the length, sequence and
conformation of such peptide probes. For instance, a
number of peptides corresponding to the immunodominant
epitopes of the gp 120 and gp 41 proteins of human
immunodeficiency virus have been selected in this way
(Richalet-Secordel et al., 1994, 1996). Recently, all-D-retro
peptides, also known as retro–inverso peptides, have been
shown to be useful as diagnostic tools (Briand et al., 1995)
and for mimicking viral epitopes (Muller et al., 1995, 1997).
In all-D-retro peptides the residues are aligned in the reverse
order of that in the parent molecule, and L-amino acids are
replaced by D-amino acids, with the result that the side-
chains have the same orientation as in the original protein
(Van Regenmortel, 1997). However, retro–inverso peptides
are much more resistant to proteolysis than classical L-
peptides (Guichard et al., 1994). The quality of the mimicry
achieved with these peptidomimetics can be easily quanti-
fied by BIAcore (Benkirane et al., 1995), and such
information has been very useful in helping to design new
synthetic peptide vaccines against foot-and-mouth disease
(Muller et al., 1997).
The biosensor technology is also particularly useful for
selecting monoclonal antibodies (Mabs) suitable for diag-
nosis. Mabs intended for use in enzyme immunoassay
should have a sufficiently slow dissociation rate constant
(about 10ÿ3 sÿ1) to prevent them from dissociating during
the washing step of the assay. The kinetic constants of the
Mabs can be determined from a few microlitres of culture
supernatant obtained during hybridoma cell culture, and
clones producing unsuitable Mabs can be discarded at an
early stage of reagent production. The binding character-
istics of antibodies can be improved by recombinant DNA
technology and site-directed mutagenesis, and biosensor
measurements can assess to what extent the binding has
been improved (Marks et al., 1992; Horenstein et al., 1994;
Malmqvist, 1994).
Diagnostic assays based on double-antibody sandwich
ELISA require pairs of Mabs that are able to bind
concurrently to the same antigen molecule, because the
epitopes they recognize are sufficiently distant from each
other. Biosensors are convenient instruments for mapping
epitopes at the surface of proteins (Fägerstam et al., 1990;
JOURNAL OF MOLECULAR RECOGNITION, VOL. 11: 163–167 (1998)
166 M. H. V. VAN REGENMORTEL ET AL.
Tosser et al., 1994; Saunal and Van Regenmortel, 1995a)
and for identifying pairs of Mabs suitable for sandwich
ELISA. It is also possible to detect the presence of
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