Available online at www.sciencedirect.com
ANALYTICAL
BIOCHEMISTRY
Analytical Biochemistry 373 (2008) 52–60
www.elsevier.com/locate/yabio
High-affinity binding measurements of antibodies to
cell-surface-expressed antigens
Palaniswami Rathanaswami *, John Babcook, Michael Gallo
Amgen Inc., Burnaby, BC, Canada V5A 1V7
Received 7 July 2007
Available online 16 August 2007
Abstract
A simple method that allows affinity measurements of antibodies to integral membrane proteins is described. Kinetic Exclusion Assay
was used to determine the concentration of free antibody that remains in solution after equilibrium has been established between the
antibody and the cell-surface-expressed antigen, from which the equilibrium dissociation constant (Kd) was determined. It eliminates
the requirement for soluble antigen and modifications such as radio-labeling or fluorescent labeling of the antibody. For one of the
cell-surface-expressed antigens, it was determined that the affinity of the antibody to the cell-surface-expressed antigen was similar to
that of the purified, soluble form of the antigen. In addition to the simplicity of the approach, the method provides a true measure
of the affinity/avidity of the antibody to the native form of cell-surface-expressed targets, including antigens that cannot be produced
in soluble forms, and to unknown cell surface antigens.
2007 Elsevier Inc. All rights reserved.
Keywords: Affinity measurement; On-cell binding; Membrane-expressed antigen; High affinity; KinExA; Cell-surface-expressed molecule; Transmembrane-expressed molecule; Human antibodies; Unmodified antibodies; Native binding
Monoclonal antibodies (mAbs)1 represent a new and
increasingly important therapeutic modality in treating
many diseases such as cancer [1,2]. When choosing the best
antibody for therapeutic purposes, in addition to specificity, the affinity of an antibody to the target is also important. A direct correlation between the affinity and the
potency of an antibody exists [3,4] and, for therapeutic purposes, antibodies with binding constants greater than
10 nM are not likely to be useful for drug targeting or
tumor imaging [5]. High-affinity mAbs, in contrast to
low-affinity mAbs, target tumors better [6] and enhance
the antibody-dependent cellular-cytotoxicity-mediated killing of tumor cells even at low antigen expression levels [7].
*
Corresponding author. Fax: +1 (604) 676 8349.
E-mail address: swamir@amgen.com (P. Rathanaswami).
1
Abbreviations used: mAbs, monoclonal antibodies; FACS, fluorescence-activated cell sorting; KinExA, Kinetic Exclusion Assay; ELISA,
enzyme-linked immunosorbent assay; NHS, N-hydroxy succinimide;
CHO, chinese hamster ovary; AgM, antigen multiplier.
0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2007.08.014
There are several established technologies/methodologies available to experimentally determine the affinity of
antibodies to soluble antigens. If the antigen is expressed
on a cell membrane, the measured affinity of the antibody
to the soluble antigen counterpart, typically the extracellular domain, may not correlate with the antibody’s binding
affinity to the native form of the antigen on the cell. In
addition, purification of membrane proteins is difficult
and may result in a loss of stability or in partially functional protein [8].
Studies characterizing the affinity measurements of antibodies to cell-surface-expressed molecules typically modify
the antibodies with fluorescent or radioactive labels so that
they can be detected [9,10]. However, it has been shown
that chemical modification of an antibody by 125I [11], flourescein isothiocyanate, and RD1 labeling can lead to binding constants that are 10 to 100-fold lower than those of
the native antibodies [12]. Some of the methods using radiolabeled antibodies require extensive cell washing after the
equilibrium is reached [10] which might lead to the dissoci-
High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
ation of the antibody from the cell membrane and disturb
the equilibrium. Bator and Reading [13] described a simple
ELISA method to measure the apparent affinity of an antibody for cell-surface antigens using Scatchard analysis.
The sensitivity of determination of the affinity by this
method is in the nanomolar range. Fluorescence-activated
cell sorting (FACS) methods also have been used to measure the affinity of cell-membrane-expressed antigens
[14,15]. However, these methods used a linear transformation of the data to calculate the affinity. Scatchard and
other linear transformations of the binding isotherm data
can sometimes conceal deviations from simple single-site
binding patterns that can be recognized by nonlinear analysis [16]. A recent report [17] describes the affinity measurement of an antibody to cell-surface receptors and
overcomes many of the above problems by using Kinetic
Exclusion Assay (KinExA). However, this reported
method requires the use of a soluble antigen as a probe
for measuring the affinity of the antibody to cell-surfaceexpressed molecules. Here we describe a modified method
in which soluble antigen is not required for measuring
the affinity of a purified antibody to its cell-membraneexpressed target.
53
ose beads were prepared by adsorbing the GST-TT-TNFa
onto glutathione–Sepharose beads.
Cell culture
CHO cells expressing a mutant noncleavable form of
TNFa on the surface of the membrane were used. Briefly,
the cDNA of TNFa was mutated at various positions to
prevent cleavage of TNFa from the surface of cells and
cloned into an expression vector. CHO cells were transfected with this mutated form of TNFa and cells expressing
TNFa in stable form on the membrane were placed under
drug selection to generate a DTNFa cell line. The cells
were cultured in standard tissue culture conditions in an
incubator with 5% CO2. Ham-F12 medium was used to
grow the cells with 10% fetal calf serum (Gibco), 2 mM
L-glutamine, 1 mM sodium pyruvate, and penicillin/streptomycin (100 IU/ml/100 mg/ml (Gibco). Cells were treated
with 8 mM sodium butyrate overnight before use.
CEM cells expressing CD45RB were cultured in RPMI
1640 medium with 2 mM L-glutamine and adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM
Hepes, and 1.0 mM sodium pyruvate. Fetal bovine serum
was added to this to a final concentration of 10% V/V.
Materials and methods
Cell-binding equilibrium assays
Azlactone beads were obtained from Sapidyne Instruments (Idaho, U.S.A). Cy5-conjugated affinity-purified
antibodies (all have minimal cross-reactivity to bovine,
horse, and mouse serum proteins) were purchased from
Jackson ImmunoResearch Laboratories, Inc. (West Grove,
PA, U.S.A). Goat anti-human Fcc fragment-specific IgG
antibody was obtained from Immunodiagnostics (Bellingham, WA, U.S.A). Protein A–Sepharose, NHS-activated
Sepharose 4 Fast Flow, and glutathione–Sepharose beads
were purchased from Amersham Biosciences, Inc. (Quebec,
Canada). ImmunoPure Fab preparation kit was purchased
from Pierce (Rockford, IL, U.S.A.). The rhTNFa was
obtained from R & D Systems (Minneapolis, MN,
U.S.A.). Human TNFa was also expressed as a fusion protein with GST and a T-cell epitope from tetanus toxin
(GST-TT-TNFa). Anti-CD45RB mAb 6G3 [18] was produced from a hybridoma. Anti-human CD45RB mAb
487 and anti-human TNFa mAb 263 were developed by
Amgen 0 s XenoMax technology [4] and produced as recombinant antibodies. Protein A–Sepharose affinity–chromatography-purified mAbs were used in all experiments.
The purity was assessed and the quantity was calculated
by nonreducing SDS–PAGE. The concentration was further confirmed by UV analysis at A280 and ELISA.
Preparation of antigen- or Ig-coated beads
Goat anti-human Fcc fragment-specific IgG antibody
(100 lg) was coupled to Azlactone beads (300 mg) as recommended by the manufacturer or to NHS-activated
Sepharose beads as described [4]. GST-TT-TNFa-Sephar-
Cells were serially diluted 1 in 2 in 11 falcon tubes using
the culture medium and containing a final concentration of
0.05% NaN3. The 12th tube contained just the medium. An
appropriate concentration of purified mAb was made in
cell culture medium and an equal volume of the mAb
was mixed with the serially diluted cells. The cells with
the mAb were then rotated at 4 C for 36 h. At the end
of 36 h, the cells were centrifuged at 2400 rpm for 4 min
and the free mAb present in the supernatant was measured
by KinExA using appropriate beads and Cy5-conjugated
anti-human secondary antibodies as described [4]. The
equilibrium dissociation constant (Kd) [17] was obtained
using KinExA software [19,20] and by ‘‘n-curve analysis’’
which fits all of the given curves to a single Kd value simultaneously [4,17].
Cell-binding affinity measurements with a corresponding Fab
fragment
The Fab fragment was prepared as per the manufacturer 0 s recommendation. The completeness of digestion
and the purity were assessed by running on an acrylamide
gel. Cells were titrated, mixed with an equal volume of Fab
fragment at defined concentrations, and continued as
described for the full mAb. The free Fab fragment present
in the supernatant was measured by KinExA using antigencoated Sepharose beads to absorb the Fab fragment and
detected using Cy5-conjugated affinity-purified goat antihuman (H+L)-specific IgG. The Kd was obtained as
described above.
54
High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
Affinity measurements with soluble TNFa and anti-human
TNFa mAb or Fab
Briefly, a constant amount of anti-human TNFa mAb
or Fab 263 was incubated with titrating concentrations of
rhTNFa antigen in sample buffer (phosphate-buffered saline with 0.1% bovine serum albumin and 0.05% NaN3).
Antigen/antibody complexes were incubated at room temperature for 36 h to allow equilibrium to be reached. The
mixture was drawn through the GST-TT-TNFa-Sepharose
beads to accumulate unbound antibody. The captured antihTNFa mAb or Fab is directly proportional to the remaining free-binding sites [4] and was detected using solutions
containing Cy5-conjugated anti-human secondary antibody in sample buffer. The Kd was determined as described
above.
Scatchard analysis
A constant number of cells was added to titrating concentrations of anti-hTNFa mAb 263 and incubated for
36 h at 4 C. At the end of 36 h, the cells were centrifuged
at 2400 rpm for 4 min and the signal generated by the free
mAb present in the supernatant was measured by KinExA
using either goat anti-human Fcc fragment-specific IgG
antibody-coupled azlactone beads or GST-TT-TNFaSepharose beads to absorb the mAb and detected using
solutions containing Cy5-conjugated affinity-purified goat
anti-human Fcc fragment-specific IgG secondary antibodies. The signal corresponding to the total antibody in each
dilution was also measured in KinExA. From the signal
obtained for the total antibody, the amount of free antibody present in the supernatant and the amount of antibody bound to cells were calculated. The Scatchard plot
was done by plotting B/F vs B [21] and the Kd was calculated from the slope.
site concentration of mAb was used as a determinant for
the KinExA software to calculate the antigen multiplier
(AgM) and the Kd using the unknown antigen method
[17]. The antigen multiplier is a multiplication constant
used in conjunction with the assumed antigen concentration (1 · 106 cells = 1 nM antigen) variable to determine
an active molar concentration of the receptor. The calculated antigen multiplication constant will be a fraction if
1 · 106 cells contain a molar concentration of receptors less
than the arbitrarily assumed concentration (1 nM). It will
be greater than one if 1 · 106 cells contain more than the
assumed antigen concentration. As shown in Fig. 1, the
Kd calculated for the anti-hCD45RB mAb 487 was 670
fM (Fig. 1B) with 95% confidence interval of 2.30 pM
(Kd high) to 140 fM (Kd low). The % error was only 2.9.
Even though the active site concentration of antibody used
in the incubation was higher than the calculated Kd, the
determined Kd bounds were reasonably narrow [4]. Based
on the bounds and the antigen concentration that binds
to 50% of the mAb (Fig. 1A), it can be concluded that
the Kd should be in the low picomolar range. An antigen
multiplier of 0.0185 was determined by the software
(Fig. 1C), which translates into the presence of 11,470 antibody-binding antigen molecules/cell as described by Xie
et al. [17].
The Kd measurements for the anti-CD45RB mouse/
human chimeric mAb 6G3 using the same CEM cells
expressing CD45RB are shown in Figs. 1D, 1E, and 1F.
The determined Kd for the mouse/human chimeric mAb
is 1.56 pM. The AgM was 0.0323, which is equal to
20,000 antibody-binding antigen molecules/cell. The variation seen in the number of receptors on the CEM cells
between mAb 487 and 6G3 may be due to the possible difference in available epitope concentration, actual differences in the active binding site concentration for each
mAb, or day to day variation of receptor expression
levels.
Results
Affinity of membrane-expressed TNFa
Affinity of CD45RB expressed on CEM cells
CEM cells expressing CD45RB antigen were harvested,
resuspended in fresh medium (2.5 · 106 cells/ml), titrated 1
in 2, incubated with a constant amount (10 pM) of antihCD45RB mAb in the presence of 0.05% NaN3, and
allowed to reach equilibrium. At equilibrium, the free
mAb left in the supernatant was measured in KinExA
using Protein A–Sepharose beads and Cy5-conjugated
affinity-purified Fab fragment of goat anti-human
(H+L)-specific IgG. The percentage free mAb left in solution was plotted against the concentration of antigen (arbitrarily defined as 1 nM of antigen per 1 million cells, see
below for more details) by the KinExA software and a sigmoidal curve was generated, as shown in Fig. 1. A theoretical curve was fit to the data (Fig. 1A) and 95% confidence
intervals were calculated by the KinExA software. The
mAb was assumed to be 100% active. The active binding
Using goat-anti human Fc-coupled beads. The DTNFaCHO cells were dissociated from plates using cell dissociation buffer. Cells (25 · 106/ml) were resuspended in culture
media, titrated 1 in 3, incubated with a constant amount
(10 or 100 pM) of anti-hTNFa mAb 263, and allowed to
reach equilibrium. After equilibrium, the free mAb left in
the supernatant was measured using goat anti-human Fcc
fragment-specific IgG antibody-coupled Azlactone beads
and the Kd was calculated by the KinExA software using
n-curve analysis [4]. A representative experiment is shown
in Fig. 2. The Kd calculated for the human anti-hTNFa
mAb 263 was 13.64 pM with Kd high of 26.8 pM and Kd
low of 7.2 pM as 95% confidence intervals. The % error
was only 2.8. AgM of 0.0828 was calculated by this method
which translates into 49,870 receptors per cell. The experiment was repeated several times and the average Kd calculated from four experiments was 18.3 pM (Table 1).
High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
55
Fig. 1. Measurement of the Kd of anti-hCD45RB mAbs for the CD45RB expressed on CEM cells. CEM cells (2.5 · 106/ml) were serially diluted and
incubated with 10 pM active binding site concentration of (A) anti-hCD45RB mAb 487 or (D) anti-CD45RB mouse/human chimeric mAb 6G3, in the
presence of 0.05% NaN3 and allowed to equilibrate. The free mAb left in the supernatant is measured as explained in the text and the % free mAb is plotted
against the antigen concentration (taking arbitrarily each million cells to be equal to 109 M antigen). The unknown antigen method was performed to
determine optimal values for Kd and the antigen multiplier. (B and E) Best fit error analysis was performed by the software for the Kd by iteratively
changing the value for the Kd while reoptimizing the other parameters of the curve fit for each iteration. The 95% confidence intervals were determined by
plotting the change in best fit for each value of Kd. (C and F) Best fit error analysis for antigen multiplier was performed similarly by iteratively changing
the value for antigen multiplier while holding the remaining parameters at their optimized values.
56
High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
Fig. 2. Determination of Kd of anti-hTNFa mAb 263 for cell-membrane-expressed TNFa. DTNFa-CHO cells (25 · 106/ml) were serially diluted and
incubated with 10 pM (triangles) or 100 pM (diamonds) active binding site concentration of anti-hTNFa mAb 263, in the presence of 0.05% NaN3, and
allowed to equilibrate. The free mAb left in the supernatant was measured as explained in the text. (A) The % free mAb is plotted against the antigen
concentration (taking arbitrarily each million cells to be equal to 109 M antigen). Multiple curve analysis using the unknown antigen method was
performed to determine optimal values for Kd and the antigen multiplier. (B and C) The 95% confidence intervals were determined by changing iteratively
the optimized value for Kd or AgM while keeping other parameters at their optimal values.
Table 1
Kd measurement of anti-hTNFa mAb 263 for TNFa expressed on the cell membrane and soluble secreted TNFa
mAb
Target
Kd (pM)
SEM
Anti-TNFa mAb 263a
Anti-TNFa mAb 263b
Anti-TNFa mAb 263c
Fab fragment of anti-TNFa mAb 263d
Fab fragment of anti-TNFa mAb 263e
Anti-TNFa mAb 263f
Anti-TNFa mAb 263g
Membrane-expressed TNFa
Membrane-expressed TNFa
Membrane-expressed TNFa
Membrane-expressed TNFa
Secreted soluble TNFa
Secreted soluble TNFa
Membrane expressed TNFa
18.4
13.4
15.9
79.0
31.6
9.1
63.3
3.14
1.05
1.75
0.05
Kd high = 42.8 Kd low = 22.8
Kd high = 13.4 Kd low = 3.5
5.45
a
b
c
d,e,f
g
Kd was measured using goat-anti human Fc-coupled beads.
Kd was measured using antigen-coated (TT-TNFa-GST-Sepharose) beads.
Average Kd was measured using both goat-anti human Fc-coupled and antigen coated beads.
Kd was determined using antigen-coated beads.
Kd was determined by Scatchard plot.
Using antigen-coated beads. Next we determined whether
there would be a difference in the calculated Kd, if antigencoated beads are used in the bead column instead of the goat
anti-human Fcc fragment-specific antibody capture.
Titrated cells were incubated with mAb as before and
allowed to reach equilibrium. After equilibrium, the free
mAb left in the supernatant was measured using GST-TTTNFa-Sepharose beads and the Kd was calculated by the
KinExA software using n-curve analysis [4]. The average
Kd calculated from four experiments was 13.4 pM (Table 1).
High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
Comparison of the affinity of full mAb with Fab. To
determine whether the on-cell binding affinity determined
for the full mAb in KinExA is the true affinity or
whether there is an avidity effect involved, the experiment was repeated using the same batch of DTNFaCHO cells, which was used in experiments with full
mAb, but replacing with Fab molecule. The cells
(20 · 106/ml) were titrated 1 in 2, incubated with a constant amount (20 or 300 pM) of Fab fragment of mAb
263, and allowed to reach equilibrium. After equilibrium,
the free Fab left in the supernatant was measured using
GST-TT-TNFa-Sepharose beads and the Kd was calculated by the KinExA software using n-curve analysis.
The antigen concentration on the cells which was determined using the full-length mAb was used as a determinant for calculating the Kd of the Fab using standard
analysis. The affinity determined for the Fab molecule
from several experiments is given in Table 1. It is clear
from this table that the affinity of the Fab to the cellsurface-expressed TNFa is about 4 to 5 · lower than
that of the full mAb, 79 pM for Fab VS 15.9 pM for
full mAb.
Comparison of affinity of full mAb and its Fab to membraneexpressed TNFa and secreted soluble TNFa
To determine whether the lower affinity of the Fab fragment of anti-hTNFa mAb 263, compared to full mAb, to
the cell-surface-expressed TNFa is due to a possible difference in the structure of the Fab or the affinity determined
for the full mAb to cell-surface-expressed TNFa may be
the result of an avidity effect, the affinities of full mAb
and the Fab were determined for secreted soluble TNFa.
A constant amount of anti-hTNFa mAb 263 (5 or 50
pM) or the Fab fragment (30 or 300 pM) was incubated
with titrating concentrations of rhTNFa antigen starting
at 100 nM and allowed to reach equilibrium and the Kd
was determined [4]. Table 1 shows that the affinity of
Fab to the secreted soluble TNFa also was about 4 times
lower than the affinity of the full mAb, 31.6 pM for Fab
VS 9.1 pM for full mAb.
Scatchard analysis of membrane-expressed TNFa
Since one conventional means of measuring affinity to a
cellular target is done by Scatchard plot, we also did Scatchard experiments. Anti-hTNFa mAb 263 (200 pM) was
titrated 1:2 in medium and incubated with 500,000 DTNFa
cells/ml for 36 h at 4 C. The free antibody left in solution
was measured and the amount of antibody bound to cells
was calculated using KinExA software and used in Scatchard analysis. Fig. 3 shows a representative experiment
and the Kd for full mAb was determined as 64 pM. The
number of antigen molecules present on the cells was calculated to be 84,300 antigens/cell. Several experiments were
done and the mean Kd was calculated to be 63.3 pM with
SEM of 5.45.
57
Fig. 3. Affinity determination of anti-hTNFa mAb 263 for cell-membrane-expressed TNFa by Scatchard analysis. Serially diluted 200 pM
anti-hTNFa mAb 263 was incubated with 0.5 · 106/ml of DTNFa-CHO
cells and allowed to reach equilibrium. The free mAb left in solution was
measured in a KinExA instrument. The data from a representative
experiment are presented as a Scatchard plot. B is the fraction of the
bound mAb and F is the concentration of the free antibody at equilibrium.
The slope of the straight line represents KA (=1/Kd) from which Kd was
calculated. The x axis intercept of the line gives the amount of mAb
bound/cell from which the binding site concentration of the receptor
(TNFa) was calculated as 84,300/cell.
Discussion
The affinity of an antibody to a soluble molecule or a
ligand can be determined easily and precisely by several
techniques such as KinExA and surface plasmon resonance
using Biacore [4,20]. When the molecule is expressed on a
cell surface, the conventional method for measuring the
cell-binding affinity of an antibody is by Scatchard analysis
[22]. This requires labeling the antibody with a radio nucleotide or some other label. Competitive inhibition methods
to measure the cell-binding affinity of mAbs, such as CNTO
95 [10], also used labeled antibody and the determined affinity ranged from nanomolar to subnanomolar. Modifications to the antibody may result in a change in the
intrinsic affinity of the antibody [11,12]. ELISA-based methods to measure the affinity of an antibody to cell-surfaceexpressed antigens albeit in the nanomolar range have been
described [13]. When the affinity of the antibody is in the low
picomolar to subpicomolar range these methods may lack
the sensitivity to measure such a high affinity. Recently,
Xie et al [17] have published an approach using KinExA
to measure the affinity of an antibody to a cell membraneantigen. Although the report describes a method to measure
the affinity of an antibody to membrane-expressed molecules without the need for labeling one of the binding components, the method still requires soluble antigen to coat
beads to measure the free antibody. For the measurement
of the affinity of an antigen present on the cell membrane,
a soluble form of the antigen for coating the beads, might
not be available, particularly in the case of integral membrane proteins with multiple transmembrane domains.
We have independently also developed a method using
KinExA to measure directly the affinity of three antibodies
to two different antigens as presented on the cell surface. In
58
High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
addition, we measured the affinity of one of the mAbs using
Scatchard analysis for comparison. The method that we
describe uses beads coated with an anti-species antibody,
which in turn captured the free antibody left in solution
and allows one to measure the affinity of the antibody as
precisely as it could have been measured by coating beads
with soluble antigen. This is especially important for membrane-bound antigens that are hard to purify or for those
antigens that lose their native confirmation after being
purified from the membrane. One can even use the Protein
A beads to capture the free antibody if the secondary detection antibody is a Fab fragment and does not contain the
Protein A binding domain. With the modifications to
KinExA technology, we measured the affinity of antibodies
to cell-membrane-expressed molecules with precision ranging from subpicomolar to low picomolar.
The binding strength of a univalent antigen to a single
combining site on a divalent IgG antibody is defined as
affinity. In solution, the binding of each combining site of
an IgG antibody to a univalent antigen is independent.
However, when the antigen is immobilized on a solid phase
or its movement is partially restricted, as on a cell membrane, epitopes on several antigens may become spatially
proximal to both IgG combining sites and the binding of
one site may increase the binding strength of the other
combining site. The sum of the strength of all binding sites
between an antibody and an antigen is defined as avidity.
Avidity is influenced by both the valence of the antibody
and the valence of the antigen. Avidity can be more than
the sum of the individual affinities. To determine whether
the cell-binding affinity measured by this method is the true
affinity or the result of an avidity effect, we measured the
affinity of the Fab fragment of one of the antibodies
(Fab molecule of the anti-hTNFa mAb 263) to DTNFaCHO cell-surface-expressed TNFa. The affinity of the
Fab to the antigen present on the cell surface was about
5· lower than that of the full mAb. Since soluble TNFa
exists as a trimer [23], it is possible that the cell-surfaceexpressed TNF is in a multimeric form and the Kd determined for the full mAb may be an avidity measurement.
This would be in contrast to the affinity of anti-hIGFR
IgG and its Fab fragment to hIGFR expressed on cells
[17]. For the anti-hIGFR mAb there was no difference in
affinity of the Fab molecule to that of bivalent IgG and
it was argued that the affinity determined was the true affinity and not due to an avidity effect. We have also shown
that the Fab, compared to the full mAb, has about 4 to
5· lower affinity to soluble TNFa. If the full-length mAb
is binding to soluble TNFa in a monovalent manner, then
the lower Fab affinity could be a result of the Fab 0 s antigen
binding site conformation being altered during its preparation. In this situation the Kd measured for the full mAb
would reflect its true affinity.
Another important finding in this study is that the affinity determined by Scatchard analysis is not the same as that
was determined using KinExA. Scatchard plots are conventionally performed by labeling the antibody and mea-
suring the cell-bound antibody after the equilibrium is
reached. With the KinExA instrument, however, we measured the free antibody left in the supernatant after equilibrium was reached and not the antibody bound to the cells.
From the measured free antibody we calculated the cellbound antibody, and used this information in the Scatchard plot. Even though we used unmodified antibody in
the experiment, the Kd calculated by Scatchard analysis
was about 4· lower (15.9 pM by KinExA vs 63.3 pM by
Scatchard) than was determined by the KinExA method.
Although close, the Scatchard result is outside the confidence interval of the KinExA result. Since there is no
appropriate way to obtain a confidence interval for the
Scatchard result, we cannot determine whether the
KinExA result falls within the uncertainty of the Scatchard
result. It was shown that the Scatchard plot introduces a
severe compression of the data at the higher concentration
end, resulting in a distortion that may affect the result [16].
In addition, it is often overlooked that measurements made
at antibody concentrations above the Kd are less influenced
by Kd than concentration, and therefore the uncertainty of
a Kd measurement is much greater [4]. Due to these reasons, the uncertainties of the Scatchard results are much
larger than that for KinExA. Scatchard experiments
attempted at lower concentrations of cells, to limit the
amount of antigen, were too noisy to analyze (data not
shown), whereas the KinExA experiments were not. With
the greater uncertainty in the Scatchard experiments, the
KinExA results are probably not in disagreement but provide a tighter, and more importantly, a defined confidence
interval. While we performed both types of experiments in
this case for comparison purposes, only the KinExA experiments resulted in precise Kd determinations.
We have often observed that proteins expressed as soluble extracellular domains do not retain their complete
native conformation compared to the full-length membrane-associated molecule. When the affinity is measured
by Biacore using soluble extracellular domains, the calculated affinity frequently does not correlate with the biological potency of the antibodies, requiring affinity
determinations to be performed by a cell binding assay.
Measuring the affinity using FACS [14,15] also might result
in irrelevant affinity measurements due to the linear transformation of the data, which may mathematically skew the
affinity. The on-cell binding affinity measurements using
KinExA potentially overcome these problems. Another
basic difference between FACS- and KinExA-based methods is that with the FACS method the antibody is titrated
with a constant number of cells and the signal of the bound
antibody corresponding to each concentration of mAb is
plotted to calculate the Kd. Additionally, a minimum number of cells are required to perform FACS analysis, which
may result in the antigen concentration being higher than
Kd leading to irrelevant results. A recent report [24]
explains how important it is to set up FACS-based experiments in a Kd-controlled fashion to obtain satisfactory
results. Moreover, with the FACS method, a significant
High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
dissociation of mAb from cells could occur during the incubation period with the secondary antibody, effecting the
equilibrium, whereas, with KinExA, the cells are titrated
with a constant amount of mAb and the free mAb present
at equilibrium is measured. This allows one to keep the
mAb at a Kd-controlled concentration. In addition, performing multiple experiments using the same amount of
titrated cells in each experiment and varying only the
mAb concentration allows one to globally analyze the data
by n-curve analysis, adding further precision to the measurement. KinExA methodology is suitable for measuring
the affinity of soluble antigens and antibodies over a broad
range (micromolar to subpicomolar) [4,25]. Here we show
that it can also be used to measure the affinity/avidity of
antibodies to cell-surface-expressed molecules, ranging
from picomolar to subpicomolar. However, if the affinity/avidity is in the nanomolar to micromolar range, this
method may warrant further optimizations. For example,
to measure a 1 nM affinity, the starting antigen concentration present on cells should be in the range of hundreds of
nanomolar to provide saturation and obtain the lower end
of the Kd curve. For cells expressing 1 · 105 antigens/cell,
to provide 100 nM of antigen, one would need approximately 6 · 108 cells/ml. This requirement would be further
exasperated if the affinity is lower than 1 nM or the number
of antigens expressed is <1 · 105/cell.
In addition to determining the affinity of antibodies to
cell-surface-expressed antigens, this method could be easily
extended to measure the affinity of a protein to its receptor
or an adaptor. However, one would need to have a sensitive method to measure the free protein left in the supernatant after the protein is equilibrated with the receptor or
adaptor present on a cell. For such determinations a pair
of sandwiching antibodies to the protein would be
required, one to couple to the bead and the other one to
use as a detecting antibody.
Monoclonal antibodies offer great promise to fulfill the
role of ‘‘magic bullets’’ in the treatment of malignancy
due to their high specificity and their ability to affect an
anti-tumor response. When the tumor antigen is expressed
in low levels on malignant cells, high-affinity antibodies
play an important role in improving the mAb potency
[7]. This report describes a sensitive method using KinExA
technology that determines the Kd of antibodies having
high affinity to membrane-associated antigens that are difficult to purify or often unknown.
Acknowledgments
We thank Steve Lackie for critical reading of the manuscript and Desiree Lim and Karen Richmond for technical
assistance.
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