q 2007 International Society for Analytical Cytology
Cytometry Part A 71A:242–250 (2007)
Analysis of Antigen-Specific Antibodies and Their
Isotypes in Experimental Malaria
Henri C. van der Heyde,1* James M. Burns,2 William P. Weidanz,3 John Horn,1
Irene Gramaglia,1 and John P. Nolan1*
1
La Jolla Bioengineering Institute, La Jolla, California 92037
Department of Microbiology and Immunology, Drexel University, Philadelphia, Pennsylvania 19129
3
Department of Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin 53706
2
Received 9 June 2006; Revision received 27 September 2006; Accepted 28 September 2006
Background: Measuring antibody production in response
to antigen exposure or vaccination is key to disease prevention and treatment. Our understanding of the mechanisms
involved in the antibody response is limited by a lack of sensitive analysis methods. We address this limitation using multiplexed microsphere arrays for the semi-quantitative analysis of antibody production in response to malaria infection.
Methods: We used microspheres as solid supports on
which to capture and analyze circulating antibodies. Antigen immobilized on beads captured antigen-specific antibodies for semi-quantitative analysis using fluorescent secondary antibodies. Anti-immunoglobulin antibodies on
beads captured specific antibody isotypes for affinity estimation using fluorescent antigen.
Results: Antigen-mediated capture of plasma antibodies
enables determination of antigen-specific antibody ‘‘titer,’’
a semi-quantitative parameter describing a convolution of
Antibodies (Ab) or immunoglobulins (Igs) are important
molecules in clearing infectious organisms from the host
but can also be used by the organism to infect phagocytic
cells. Thus, assessing the levels of Abs against infectious
agents is fundamental to analyzing (1) the immune
response to the infectious organism, and (2) assessing
whether a vaccination regime is effective in eliciting antigen-specific antibodies. The light and heavy chains of an Ab
molecule are crosslinked by disulphide bonds, and two
pairs of light and heavy chains comprise an intact Ab. The
variable domains of the heavy and light chains determine
the Ab’s specificity and bind the antigen. The constant or
Fc region of the heavy chain (g[1, 2a/c, 2b, 3c, and 3], l, a,
d, and e) expressed defines the isotype of the antibody
(IgG[1, 2a/c, 2b, and 3], M, A, D, or E) and its functions
because the Fc region binds to specific Fc receptors or to
complement molecules. The contribution of the light chain
constant regions (j and k) to antibody function remains to
be elucidated. Because the function of the antibody is
determined in part by its isotype, assessing the levels of
each Ab isotype is also an important parameter for defining
the protective immune response and effective vaccination.
antibody abundance and avidity, as well as parameters
describing numbers of antibodies bound/bead at saturation and the plasma concentration-dependent approach to
saturation. Results were identical in single-plex and multiplex assays, and in qualitative agreement with similar parameters derived from ELISA-based assays. Isotype-specific
antibody-mediated capture of plasma antibodies allowed
the estimation of the affinity of antibody for antigen.
Conclusion: Analysis of antibody responses using microspheres and flow cytometry offer significant advantages in
speed, sample size, and quantification over standard
ELISA-based titer methods. q 2007 International Society for Analytical Cytology
Key terms: immune response; Plasmodium; AMA1;
MSP1; affinity
Measurement of the levels of antigen-specific antibody
is often performed using an enzyme-linked immunoassay
(ELISA) format, wherein antigen is coated non-specifically
onto wells of plates. This antigen then captures the specific antibody in the sera or plasma. After washing the
unbound antibody from each well, a secondary antibody
specific for the captured antibody is added. This secondary antibody is generally conjugated with an enzyme that
cleaves a colorimetric substrate, and the signal is detected
spectrophotometrically. Although affinity-purified antigen
specific antibodies can serve as a quantitative standard in
such assays (1), most often results are expressed in arbitrary units relative to a reference serum (2) or protein (3)
for semi-quantitative analysis. The standard ELISA assay
Grant sponsor: NIH; Grant numbers: AI40667, AI12710, AI49585, and
EB003824.
*Correspondence to: Henri C. van der Heyde or John P. Nolan, La Jolla
Bioengineering Institute, 505 Coast Boulevard, La Jolla, CA 92037, USA.
E-mail: [email protected]
Published online 24 January 2007 in Wiley InterScience (www.
interscience.wiley.com).
DOI: 10.1002/cyto.a.20377
ANTIGEN-SPECIFIC ISOTYPE LEVELS
has limitations in that it can only measure the levels of
antibody specific for a single antigen and only one isotype
per well. Because the ELISA is an enzymatic process, the
time of development of the assay is critical and can result
in significant plate to plate variation. The ELISA assay is
generally performed in 96 well plates, and typically
requires a minimum of 0.25 lg of antigen to coat the well
and 50 ll of sample per assay point.
Recently, microsphere-based flow cytometry (4) has
been used to adapt these assays for more efficient analysis.
In this approach (5–7), antigen is conjugated onto microspheres, which in turn are used to capture the antigenspecific antibody present in the serum or plasma. The captured antigen-specific antibody is then detected by a secondary fluorescence-labeled antibody specific for the antibody isotype of interest. The median intensity of fluorescence on the beads is assessed by flow cytometry and is a
measure of the amount of plasma antibody bound to the
beads. This approach has similar or improved sensitivity
over the ELISA assay, requires less antigen and sample
than an ELISA, and can be multiplexed to measure the
levels of several antigens or isotypes simultaneously. However, like an ELISA, this approach typically reports the
amount of antigen binding activity present in relative
terms in the form of a titer, which is a convolution of antibody concentration and affinity.
To begin to address these issues of antibody abundance
and affinity, we have extended this microsphere-based
approach to more directly measure these quantities in the
analysis of the immune response to malaria infection.
Malaria is a major health problem for nearly a third of the
world’s population living in areas of endemic transmission
where the disease is characterized by high mortality especially in children less than 5 years of age (8). We selected
experimental infection of mice with Plasmodium chabaudi as our model system because (i) antibodies are
required to sterilize this infection (9–11) and (ii) protein
antigens have been identified that confer protection from
experimental malaria upon vaccination (12). Plasmodium
falciparum apical membrane antigen-1 (Pf AMA-1) and
merozoite surface protein-1 (PfMSP-1) are leading human
malaria vaccine candidates; the murine analogues, P. chabaudi apical membrane antigen-1 (Pc AMA-1) and merozoite surface protein-1 (PcMSP-1) exhibit marked protection upon vaccination in experimental P. chabaudi
malaria (13). We studied the antibody response to these
two antigens following infection and vaccination.
MATERIALS AND METHODS
Infection and Vaccination of Mice
P. chabaudi adami, stored as a frozen stabilate, was
injected into source mice to generate an inoculum for the
experiments. C57BL/6 female mice were obtained at 4–5
weeks of age from Jackson Laboratories (Bar Harbor, ME),
housed in microisolator cages and provided food and
water ad libitum. The mice were injected with 1 3 106
P. chabaudi parasitized erythrocytes i.p. when they were
between 6 and 15 weeks of age. Parasitemia was assessed
Cytometry Part A DOI 10.1002/cyto.a
243
by counting the number of infected erythrocytes among
200 and 1,000 erythrocytes in Giemsa-stained thin films of
tail blood. After resolving the initial infection (day 65 postinfection), the mice were injected i.p. with 1 3 107 P. chabaudi parasitized erythrocytes. Fifteen days after the second P. chabaudi infection, the animals were anesthetized
with ketamine (120 mg/Kg) and Xylazine (10 mg/Kg), and
the blood obtained by cardiac puncture. The blood was
centrifuged at 600g for 20 min to remove cells and the
plasma was aliquoted and stored at 280°C.
Groups of mice were vaccinated with a fragment of Pc
MSP-142 as described in detail in Ref. 13. Briefly, C57BL/6
mice were immunized subcutaneously with 25 lg of
recombinant PcMSP-1 with 25 lg of QuilA (Accurate Scientific, Westbury, NY) as the adjuvant. Three weeks after
the primary vaccination, the animals were boosted with
the identical dose of PcMSP-1 in adjuvant. Ten days later,
blood was obtained via the retro-orbital plexus from
anesthetized mice and processed into sera. The Institutional Animal Use Committees of University of WisconsinMadison, Drexel University, and La Jolla Bioengineering
Institute approved all procedures.
Conjugation of Antibodies and Antigens
to Microspheres
Two distinct sizes of microspheres were purchased from
Bangs Laboratories (4.5 and 5.4 lm; Fisher, IN) and conjugated with Pc MSP-1 and Pc AMA-1 respectively using a single step carbodiimide chemistry as detailed in Ref. 14.
Briefly, 0.1 mg of the selected protein was incubated with
1 3 107 microspheres for 1 h (total volume 0.1 ml). The
volume was increased to 1 ml of phosphate buffered saline
(PBS), and then 10 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride was added. The mixture
was kept at 4°C for 1 h. The microspheres were washed
three times with 1 ml of PBS with 0.02% Tween-20 and
resuspended at a concentration of 1 3 107/ml.
Assessment of Antigen-Specific Plasma or Serum
Antibodies Using Antigen-Coated Micropsheres
The general approach is outlined in Figure 1A. Threefold dilutions (10 ll) of the plasma or serum with initial
dilution of 1:10 was added to 1 3 105 beads (10 ll) in PBS
containing 10% control serum and 0.2% bovine serum albumin. The control plasma or serum was matched to the
species of the detecting antibody. The mixture was incubated at 4°C for 2 h with shaking. The beads were washed
twice in PBS with 0.2% BSA (PBS/BSA) using a microfilter
plate (Millipore, Billerica, MA) and vacuum. Thirty microliters of detecting antibody was added at a concentration
of 33 lg/ml and incubated at 4°C for 0.5 h with shaking.
The detecting antibodies were: goat anti-mouse Ig (Alexa
488-conjugated; Invitrogen, Carlsbad, CA), goat antimouse IgG1 (Alexa-488, Invitrogen), rat anti-mouse IgMphycoerythrin (-PE), rat anti-mouse IgA-FITC, rat antimouse IgE-biotinylated (and stained with streptavidin-PE,
Molecular Probes/Invitrogen), rat anti-mouse IgG2a-FITC,
rat anti-mouse IgG2b-FITC, and rat anti-mouse IgG3-FITC.
244
VAN DER HEYDE ET AL.
FIG. 1. Schematic diagram of assays used in this study. (A) Measurement of plasma Ig binding to immobilized antigen involves preparation of antigen-bearing capture beads, capture of antigen-specific antibody from plasma, and detection using fluorescence-labeled secondary antibodies. (B) Measurement of
antigen binding to captured plasma antibodies involves preparation of isotype-specific antibody-bearing capture beads, capture of specific antibody isotypes from plasma, and measurement of binding to fluorescent antigen.
All rat mAbs were obtained from Pharmingen, San Diego,
CA. The microspheres were washed three times with PBS/
BSA and then transferred to tubes for analysis by flow
cytometry. The microsphere mean fluorescence intensity
was plotted as a function of plasma dilution, and the titer
determined as the highest dilution that exceeded a threshold five standard deviations above the background (15).
Assessment of Antigen Binding to Immobilized
Plasma Antibody
The general approach is outlined in Figure 1B. The affinity of plasma antibody for antigen was assessed by capturing antibody from plasma onto anti IgG-coated beads (prepared as for antigen-coated beads above) and titrating fluorescence-labeled antigen. Beads (20 ll at 1 3 106/ml)
bearing anti-IgG were incubated with plasma (20 ll of a
1:10 dilution) for 1 h on ice, then washed twice and resuspended in 20 ll. The beads (1 ll) bearing captured antibody were then incubated with increasing amounts of fluorescein-labeled AMA-1 in a total volume of 20 ll for 30 min
on ice, then diluted to 500 ll with PBS and measured by
flow cytometry. In preliminary experiments, we demonstrated that equivalent results obtained in a duplex assay
involving two different sized beads gave equivalent results
to each antigen analyzed individually (data not presented).
Calibration of Flow Cytometry Measurements and
Estimation of Binding Parameters
The microsphere mean fluorescence intensity (MFI,
which ranged from 1 to 6,000) of samples was converted
to units of mean equivalent soluble fluorophores (MESF)
using Quantum-FITC and Quantum-PE MESF calibration
microspheres (Bangs Laboratories) and linear regression.
The antibody to fluorophore to protein ratios were determined by absorbance, essentially as described in Ref. 16.
The relative quantum yield (Qr) of fluorescein conjugated to
antibodies compared with free fluorescein was determined
using absorbance calibrated solutions of antibodies and free
fluorescein measured in a SPEX fluorimeter. The F/P and Qr
were used to convert MESF values to molecules of antibody
bound per microsphere. The Qr of Alexa488 relative to fluorescein was measured by comparing the fluorescence of
antibodies labeled with each bound to identical microspheres. The results from PE-conjugated antibodies were not
corrected for F/P or Qr and are presented as MESF values.
Antibody and antigen bead binding data was fit with an equation describing a hyperbolic single site binding curve:
f ¼ ax=ðb þ xÞ
ð1Þ
where a equals the fluorescence signal at saturation and b
equals the equilibrium dissociation constant that describes
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ANTIGEN-SPECIFIC ISOTYPE LEVELS
245
the approach to saturation. For binding of plasma or serum
antibodies to antigen-coated beads, b is expressed in units
of reciprocal dilution, since the absolute molar concentration of antibody present is not known. For binding of fluorescent antigen to beads bearing plasma or serum antibodies, b has units of M.
To estimate the concentration of antigen-specific IgG1, we
multiplied the affinity, measured by the titration of labeled
antigen binding against captured plasma antibody, by the
dilution factor that gave half maximal binding of plasma antibody to antigen coated beads to calculate the concentration
of antigen specific antibody in the original plasma sample.
ELISA-Based Analysis of Antigen-Specific
Plasma Antibodies
The ELISA assay to measure the levels of antigen-specific
antibodies and their isotypes in the immunized mice were
performed as described previously (3). The wells of high
protein binding ELISA plates (Corning-Costar, Cambridge,
MA) were coated with PcMSP-1 protein (25 lg in 200 ll
100 mM NaHCO3/Na2CO3 buffer, pH 9.3). After blocking
the wells with 5% non-fat milk powder, duplicate samples
of serum (50 ll) were added to antigen coated wells for 2
hours at dilutions of 1:200, 1:1,000, 1:10,000, 1:100,000,
and 1:1,000,000 to obtain values in the linear range of the
normalization curve. The normalization curve was
obtained from wells coated with dilutions ranging from 16
ng/ml to 1mg/ml of purified myeloma proteins (Zymed,
San Francisco, CA). Antigen-specific antibodies were
detected with horseradish peroxidase-conjugated rabbit
anti-mouse IgG1, G2b, G3 (Zymed), or goat anti-mouse
IgG2a b allotype (Southern Biotechnology Associates, Birmingham, AL) and 2,20-azinobis(3-ethylbenz-thiazolinesulfonic acid) as substrate. The absorbance was read at 405
nm and values between 0.1 and 1 were considered within
the linear range of the instrument.
Statistical Analysis and Curve Fitting
Analysis of variance (ANOVA) with the StatviewTM program (SAS Institute, Cary, NC) with Fisher’s post-hoc test
was performed to statistically compare all measurements
with a P-value cut-off of 0.05. The mean and standard
deviation of the results are reported in text and figures.
RESULTS
Assessment of the Levels of Total Ab Specific for
Either MSP-1 or AMA-1 in Plasma of
Infected Mice
To determine whether the total Ab titers specific for
selected Plasmodium antigens are measurable by a microsphere-based assay, we incubated microspheres conjugated with PcMSP-1 or PcAMA-1 for 2 h with a serial dilution of immune plasma or with plasma from an uninfected
mouse to capture the antigen-specific antibody on the
microspheres (Fig. 1A). The microspheres were then
washed and fluorescence labeled with goat anti-mouse IgAlexa 488. For both MSP-1 and AMA-conjugated beads,
background staining at low concentrations of plasma was
Cytometry Part A DOI 10.1002/cyto.a
FIG. 2. Measurement of total antigen-specific Ig antibody in plasma.
Antigen-specific plasma antibodies from infected or uninfected mice were
captured onto antigen-coated beads, and detected with fluorescent antiimmunoglobulin antibody. (A) Semi-log plot. (B) Linear plot.
negligible, and fluorescence increased with increasing
plasma concentration in a characteristic sigmoidal fashion
on a semi-log plot (Fig. 2A). Approximately 2.16 3 106
and 1.33 3 106 FITC-anti-Ig molecules were bound per
bead at the highest concentration of plasma (1:20 dilution) for PcMSP-1 and PcAMA-1 beads respectively, likely
reflecting a difference in antigen density on the beads.
Staining of beads exposed to plasma from uninfected mice
was very low. Each data point represents the geometric
mean of approximately 500 events for which the coefficient of variation (CV) was 30%. The variation in these
means between replicates of the same dilution of the
infected plasma is small (CV 3%; see error bars in Fig.
2A), indicating that replicates are not required for accurate assessment of the levels of antigen-specific antibody.
An antibody titer was defined as the lowest dilution that
gave a positive signal above a background-defined threshold (Table 1). As an alternative analysis, when displayed
with a linear x axis (Fig. 2B) the binding data have the familiar hyperbolic shape of a simple reversible binding
interaction and is well fit by the equation describing a hyperbola, which returns parameters that describe the number of antibody binding sites and the concentration-dependent approach to saturation (Table 1).
VAN DER HEYDE ET AL.
246
Table 1
Analysis of Anti-AMA-1 and Anti-MSP-1 Antibodies in the Plasma of P. chabaudi-Infected Mice
Isotype
Titera
AMA
Antibody binding
sites per beadb
Ig
IgA
IgE
IgM
IgG1
IgG2a/c
IgG2b
IgG3
7.6 3 1026
1.9 3 1023
ND
8.9 3 1025
6.2 3 1024
2.3 3 1025
6.9 3 1025
ND
1.33 3 106
nd
nd
5.08 3 104
8.19 3 105
1.14 3 106
4.02 3 106
nd
Antigen binding
activityc
3.90 3 1023
nd
nd
4.9 3 1023
6.21 3 1023
4.3 3 1023
3.9 3 1022
nd
Titera
MSP
Antibody binding
sites per beadb
Antigen binding
activityc
2.28 3 1025
5.6 3 1023
ND
2.1 3 1024
6.9 3 1025
2.3 3 1025
6.9 3 1025
5.6 3 1023
2.16 3 106
nd
nd
3.12 3 104
2.56 3 106
2.02 3 106
2.77 3 106
nd
5.7 3 1023
nd
nd
1.02 3 1022
3.64 3 1023
9.85 3 1023
1.86 3 1022
nd
a
Calculated as the lowest dilution above background as described in Methods.
Calculated from curve fit as described in Methods in units of molecules per bead, except for IgM (MESF per bead).
c
Calculated from curve fit as described in Methods, units of dilution.
nd, not determined; ND, not detected.
b
Assessment of Activity of Ab Isotypes Specific for
Either MSP-1 or AMA-1 in Plasma of Infected
Mice by MFA
The assay described above for total antigen-specific Ab
was repeated using isotype specific antibodies rather than
total Ig-specific detection antibody for both MSP-1 and
AMA-1 conjugated beads (Fig. 3). These results indicate
there were differences in the titers of isotypes for AMA1versus MSP-1-specific antibody in the immune mouse (Table 1). PcMSP-1-specific antibody with the IgM, G1, G2a/c,
and 2b were readily detected, whereas little if any PcMSP1-specific antibody of the IgA and G3 isotypes was
detected (data not presented).
FIG. 3. Measurement of antigen-specific antibody isotypes. Antigen-specific plasma antibodies from infected or uninfected mice were captured onto antigencoated beads, and detected with fluorescent isotype-specific anti-immunoglobulin antibodies. (A) anti-IgM. (B) anti-IgG1. (C) anti-IgG2a/c. (D) anti-IgG2b.
Cytometry Part A DOI 10.1002/cyto.a
ANTIGEN-SPECIFIC ISOTYPE LEVELS
247
and polyclonal antibodies used, the epitopes, concentrations and affinities of antibodies in plasma, and competition
among these for binding to antigen on the bead. In addition
there are assumptions in comparing FITC and Alexa488 fluorescence intensities and in the valency of binding that
introduce uncertainty into estimates of the absolute numbers of plasma antibodies bound. Finally, as for the ELISA,
the titer value might be expected to depend on the antigen
concentration, which in the bead assay would be affected
by bead size, antigen density, and bead number. However, if
these parameters differ by less than a factor of two, as is the
case for the data presented here, the impact on the titer estimate from the logarithmic x axis would be minimal. For all
of these reasons, the titer-based estimates of binding activity
must be taken as semiquantitative.
Comparison of the Assessment of Activity of Ab
Isotypes Specific for MSP-1 and AMA-1 in Plasma of
Infected Mice by MFA and ELISA
FIG. 4. Antibody responses to vaccination with MSP1. Serum from
vaccinated mice were analyzed using a ELISA (A) or microsphere-based
(B) methods.
While it is common to normalize such titration data, we
have chosen to present the data calibrated in numbers of
antibody molecules bound per bead. In principle, one might
expect the sum of binding of the individual isotypes in Table
1 to equal the total Ig, several factors complicate such a
straightforward analysis including uncertainities in the specificities and cross reactivities of the various monoclonal
To compare the ELISA assay to the microsphere-based
assay, we analyzed the same serum sample from a mouse
vaccinated with PcMSP-1 by both techniques. Presented in
Figure 4A are the optical densities resulting from development of HRP-conjugated isotype-specific anti-IgG antibodies measured from a serum dilution series in wells coated
with 0.25 lg of PcMSP-1. A titer can estimated by setting a
threshold for positivity (Table 2), but the reliability of this
parameter is compromised by variation in the activities of
the enzyme conjugate among the isotype specific antibodies. To account for this serial dilutions of selected myeloma proteins (IgG1, 2a/c, and 2b) adsorbed to microwells serve as standards and are used to normalize the data
(3). Data points from the serum dilution with ODs greater
than 0.1, but within the linear range of the myeloma protein normalization curve (OD < 1.0), are converted to a
Unit value that accounts for differences in reporter enzyme
activity, if it is assumed that adsorption of the different myeloma proteins to the microwells is uniform. The limited
dynamic range of the colorimetric readout restricts measurement of serum antibody binding to 4 two-fold dilutions (16-fold range of concentrations) at the lower end
of the titration curve. There were modest differences
between the ratios of the isotype ratios determined by titer
Table 2
Analysis of Anti MSP-1 Antibodies in the Serum of P. chabaudi-Infected Mice
Isotype
Ig
IgG1
IgG2a/c
IgG2b
IgG3
Microsphere
Antibody binding sites per beadb
Titera
25
6.9 3 10
6.9 3 1025
6.9 3 1025
6.9 3 1025
nd
a
6
1.95 3 10
1.98 3 106
1.46 3 106
1.56 3 105
nd
ELISA
Antigen binding activityc
24
4.69 3 10
3.40 3 1023
3.16 3 1023
3.29 3 1023
nd
Calculated as the lowest dilution above background as described in Methods.
Calculated from curve fit as described in Methods in units of molecules per bead.
Calculated from curve fit as described in Methods, units of dilution.
d
Calculated from a standard curve of adsorbed myeloma proteins as described in Methods.
nd, not determined.
b
c
Cytometry Part A DOI 10.1002/cyto.a
Titera
Units/mld
nd
2.2 3 1026
2.6 3 1022
2.6 3 1022
nd
4.34 3 104
8.92 3 103
3.32 3 103
248
VAN DER HEYDE ET AL.
(dilution at OD 5 0.1) with ratios determined by the Unit
value; for titer: IgG1 10 3 IgG2a/c 10 3 IgG2b and
Unit value: IgG1 53 IgG2a/c 10 3 IgG2b, where 3
represents the approximate fold increase in titer.
The microsphere-based assay was used to assess the
same serum from the PcMSP-1-immunized mouse. As for
the ELISA, a plot of fluorescence vs dilution allows estimation of a threshold crossing-based titer. In this case, differences in reporter fluorescence of the different isotype-specific antibodies can be directly measured and combined
with the calibrated flow cytometry measurement to
express data as molecules of reporter antibody bound per
microsphere (Fig. 4B). Thus, a separate normalization procedure is not required. Moreover, the larger dynamic range
of the fluorescence measurement enables measurement of
serum antibody binding over 9 three-fold dilutions
(20,000 fold range of concentrations), allowing analysis
of undiluted (save for reagents) serum. The rank order of titer was similar between the two members, with the exception of IgG2b, which was higher for the ELISA methods.
The most likely explanation of this is differences in the
specificity of antibodies used (rabbit polyclonal for ELISA,
rat monoclonal for the bead-based assay). Beyond the qualitative rank order comparison, while each method may be
useful for relative estimates of antigen binding activity,
cross-platform comparisons are not justified, as the ‘‘titer’’
value will be sensitive to differences in antigen concentration, sample volumes, signal amplification, and reagents.
Estimation of Composite Affinity of Antibody
The results described above demonstrate that the
microsphere-based approach to measuring antigen capture of plasma antibodies offers several advantages over
the ELISA format for relative measure of plasma or serum
antigen binding activity, as has been reported by others
(5,17,18). Beyond increased sensitivity, precision, and efficiency however, microsphere-based flow cytometry provides an opportunity to deconvolve the contributions of
plasma antibody concentration and affinity to the measured titer. The antigen-antibody interactions of interest
can be viewed as a simple bimolecular binding interaction, and the binding of antibody to immobilized antigen
can be presented as a function of plasma concentration
(Fig. 5A), rather than as an inverse concentration (dilution) as is done in a titer determination. In this presentation, the binding curve has the familiar hyberbolic shape
of a specific bimolecular binding interaction, and is well
fit by the equation describing a hyberbola (Eq. 1). In this
analysis, Bmax represents the total amount of antigen-antibody complex on the bead at saturation, and the KD term
estimates the concentration of antibody that gives half
maximal binding. Because the absolute concentration of
plasma antibody is not known, like the titer this term is a
convolution of antibody concentration and affinity.
To measure the affinity of plasma antibody for antigen,
we used an IgG1 isotype-specific antibody to capture
plasma IgG1 antibody onto microspheres, and titrated
these plasma antibody-coated beads with fluorescence-la-
FIG. 5. Estimation of antigen-specific antibody affinity and concentration from infected mice. (A) Binding of plasma antibody to AMA1-coated
beads. Data of Figure 3B replotted. (B) Measurement of affinity of fluorescent antigen for captured plasma antibody.
beled antigen (Fig. 1B). Presented in Figure 5B is the binding of fluorescent-AMA-1 to beads bearing captured
plasma IgG1 antibody. Binding was specific and saturable,
and non-specific binding to beads incubated with plasma
from uninfected mice was low. A fit of these data to Eq. 1
gave a KD estimate of 42 nM. With the KD estimate in
hand, it is then possible to revisit the data in Figure 5, and
use this value, together with the reciprocal dilution value
to calculate a plasma IgG1 PcAMA-1-specific antibody concentration of 6.8 lM (or 1.0 ng/ml). This calculation
does not account for the effect of competition among antibody types and isotypes in the antigen-based capture assay
(Fig. 1A), a common feature of any immobilized antigenbased capture assay, and thus likely slightly underestimates the actual concentration of MSP-1 specific IgG1.
DISCUSSION
Assessing the concentration and affinity of Ag-specific
Ab elicited by infection or immunization is not trivial for a
number of reasons. First, there is not a single Ab molecule
identical at the sequence level that binds to the Ag; rather,
the selection process of different Ab-producing cells in
the germinal centers leads to a population of different Ab
molecules, individual types of which likely have different
Cytometry Part A DOI 10.1002/cyto.a
ANTIGEN-SPECIFIC ISOTYPE LEVELS
abundances in plasma and affinities for the Ag. The Abproducing cells, and thus the number and affinities of the
antibody sub-populations present in plasma, can change
over time during the evolution of the immune response.
This complexity has implications for the different approaches for assessing antigen-specific antibodies in plasma.
The classic test is to immobilize antibody on a surface (typically a microwell bottom in an ELISA format), capture antigen-specific antibody with the immobilized antigen, and
then detect the captured antigen-specific antibody with a
labeled type- or isotype-specific secondary antibody. The
amount of the secondary antibody retained on the surface
is the measured, and is assumed to reflect the amount of
the antigen-specific Ab present in the plasma sample. However, the amount of a particular antigen-plasma Ab-secondary Ab complex formed can also depend on the distribution
of different types or isotypes of antigen-specific Abs in
plasma, as well as their affinities for the antigen. Thus, the
signal measured in this type of assay represents the results
of a competition for antigen by a number of populations of
Abs that vary in abundance and affinity.
Limitations of the assay methods used for assessing Agspecific Abs make the deconvolution of Ab abundance and
affinity in such an assay nearly intractable. A major issue is
the calibration and normalization of assay data. This is complicated by variations in the amount of immobilized antigen
on the assay surface, and differences in the labeling characteristics of the secondary antibodies, both of which can
result in differences in measured signal that are not related
to the amount (or affinity) of Ab in the sample. One
approach designed to address this is to construct a ‘‘standard curve’’ by immobilizing different amounts of individual
antibody isotypes on the surface of different microwells,
and use these to capture the labeled secondary Ab. Linear
ranges of signal from these wells are compared from wells
containing Ab-plasma Ab-secondary Ab complex, and used
to express sample data in units of equivalent mass of type
or isotype-specific Ab (Fig. 4). This approach addresses differences in reporter activity of the different secondary Abs,
but introduces uncertainties in variation in the well to well
immobilization of the ‘‘standard’’ Abs in the first place, and
does not address the well to well variation of antigen, nor
the competition among plasma Abs for that Ag. McHugh
(7) used this approach with microspheres for quantifying
detection of anti HCV IgG, but not isotypes. Another
approach prepares a standard preparation of antigen-specific immune serum that is then used as a standard to compare results from different experiments, allowing expression of the results in arbitrary units (eg, milliMerck units
(5). This approach will control for well to well variations in
immobilized Ag, but provides results in arbitrary, rather
than absolute, units and does not address different labeling
of secondary Abs, making it difficult to assess Ab types and
subtypes.
The approach presented here adapts the ELISA-based
assay to microspheres. By preparing a single lot of Agcoated microspheres that is used to measure all samples,
issues of sample to sample variation in immobilized Ag is
controlled, resulting in excellent measurement precision
Cytometry Part A DOI 10.1002/cyto.a
249
(Fig. 1). Because the labeling efficiency (the F/P and Qr)
of the secondary reporter Abs can be directly measured, it
is possible to directly compare the signals arising from different isotype-specific secondary antibodies. Moreover,
because the response of the flow cytometer can be calibrated in terms of mean equivalent soluble fluorophores
(MESFs), the microsphere fluorescence values can be
expressed in absolute terms of numbers of molecules of
secondary Ab per microsphere over more than three
orders of magnitude of signal dynamic range. These features of quantification, coupled with the very small sample volumes required (>20 ll) and the ability to assess the
response to multiple antigens simultaneously, provide the
microsphere-based flow cytometry approach with several
important advantages over the ELISA-based formats.
However, these mechanical improvements still leave us
with the issue of how to quantify the abundance and affinity of the different Ab populations present in a plasma
sample. The familiar plot of signal vs log dilution and the
traditionally derived ‘‘titer’’ value does not provide a direct
way to address these issues. A different approach is
derived from the analysis of binding interactions, and displays the data as a plot of signal intensity vs volume of
sample (Fig. 5B). This curve has the familiar hyberbolic
shape of an equilibrium binding curve for reversible molecular interactions, of which the Ag-Ab interaction measured here is one. The data are well fit by the equation for
a hyperbola, and parameters can be estimated that
describe the saturation value and the approach to that
saturation. In a simple reversible biomolecular interaction
between a known concentrations of soluble ligand and an
immobilized receptor, these parameters would correspond to the total receptor number on the bead and the
equilibrium dissociation constant (KD) of the complex. In
the present case, the total number of antigen molecules
(the receptor) on the microsphere can be estimated, and
the saturation value for each Ab type or isotype (revealed
by the staining with the labeled secondary Ab) reflects the
competition among the various Ab sub-populations within
plasma for the Ab binding sites (the immobilized antigen.
The concentration of the soluble ligand (the antigen-specific plasma Ab) is not known, so the parameter that
describes the approach to saturation (which has units of
volume) is a convolution of concentration and affinity for
each of the Ab types and sub-types, assessed in a situation
where there is competition among antibody populations
in plasma for the immobilized antigen. Thus the parameters derived from this analysis to describe the antigen-specific Abs in plasma still do not give fundamental constants,
but rather, like the ‘‘titer’’ value represent a composite
value that reflects several factors related to abundance
and affinity. However, these parameters are more directly
related to fundamental binding constants than are titer
values, and have the advantage of allowing quantitative
comparison of Ab types and sub-types.
To deconvolve the Ab affinity and concentration, more
information is needed. If we know the concentration of
each Ab sub-type within plasma, it is then straightforward
to estimate the affinity. However, it is very difficult to deter-
250
VAN DER HEYDE ET AL.
mine antigen-specific Ab concentrations using available
methods. It is however possible to estimate affinity by
measuring antigen binding to antibody. In this approach,
anti-immunoglobulin antibody-coated beads are mixed with
plasma to capture plasma antibodies on to the surface of
the microspheres. The plasma antibody-bearing microspheres are then titrated with increasing concentrations of
fluorescence-labeled antigen, and the binding curve analyzed to estimate the affinity (KD) of the antigen-antibody
interaction. In the characterization of antibodies, which are
multivalent, the term avidity is often used in place of affinity, in recognition that the monovalent affinity is often
obscured by the multivalency of a ligand. This is especially
appropriate when measuring the binding of an antibody to
an antigen bearing surface (such as microwell or microsphere surface, or to the surface of a cell membrane) where
one antibody may engage two antigen molecules and thus
bind more tightly than if the binding to the surface were
monovalent. In this case (Fig. 5B), antibody is immobilized
on the surface and soluble antigen is binding to antibody.
Assuming there is no steric or other factors interfering with
binding the two IgG antigen binding sites on each IgG molecule, this measures a monovalent interaction, and thus the
more precise term affinity is appropriate.
This affinity estimate represents the average affinity of
all of the IgG1 isotype antibodies in plasma. Although this
is a polyclonal population of antibody molecules with affinities that may be somewhat different, the data are welldescribed by a single site model, indicating that the affinities of the different monoclonal species are comparable.
Knowledge of this affinity then allows one to calculate the
concentration of antigen specific plasma antibody using
the plasma titration data. The plasma titration data is complicated by the fact that although only one antibody type
or isotype is being measured, all antigen-specific antibodies present are competing for antigen on the bead. This,
effect, which is common to any immobilized antigenbased capture assay, will result in a slight underestimate of
the actual concentration of a particular isotype.
The development of antigen-specific antibody responses
is poorly understood, especially in terms of the amounts
and affinities of antibodies produced. The traditional
approach of measuring an antibody titer does not allow
these factors to be distinguished. Here, using antigen and
antibody isotype-specific reagents, we demonstrate that it
is possible to measure antigen-specific antibody affinity
directly, and use this to deconvolve antigen-specific antibody concentration from plasma titer data. This approach
sheds a newly quantitative light onto the development of
antigen-specific antibody development.
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Cytometry Part A DOI 10.1002/cyto.a