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Clinical and Diagnostic Laboratory Immunology, November 1999, p. 977-980, Vol. 6, No. 6
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Relative Avidities of Human Immunoglobulin G
Antibodies for Streptococcal Pyrogenic Exotoxins A and B
Ellen M.
Mascini,*
Margriet
Jansze,
Jan
Verhoef, and
Hans
van Dijk
Eijkman-Winkler Institute for Microbiology,
Infectious Diseases, and Inflammation, University Medical Center,
Utrecht, The Netherlands
Received 19 February 1999/Returned for modification 21 April
1999/Accepted 1 September 1999
 |
ABSTRACT |
In this pilot study, we investigated the relative avidities for
streptococcal pyrogenic exotoxin A (SPE-A) and SPE-B of antibodies in
sera from patients with fatal streptococcal toxic shock-like syndrome
and from healthy individuals and in intravenous immunoglobulin (IVIG)
preparations. We observed a great variation in the relative avidities
of patient, control, and IVIG immunoglobulin G (IgG) (values estimated
to be between 10
7 and 1011 M), with mean
values for patient IgG about 10-fold lower than those of control IgG.
| |
TEXT |
Group A streptococci (GAS) are a
common cause of a wide variety of mild human infections, including
pharyngitis, scarlet fever, and erysipelas. In addition, GAS are
sometimes involved in life-threatening diseases like streptococcal
toxic shock-like syndrome (TSS) and necrotizing fasciitis. Since the
mid-1980s, outbreaks of severe GAS infections have been reported in
various parts of the world (1, 14). Despite prompt
antibiotic treatment, the mortality rate in patients with these
syndromes remains high (30 to 80%) (1, 14). Predisposing
conditions such as advanced age, diabetes, malignancy, and
immunosuppression have been recognized but, remarkably, a substantial
proportion of patients consists of previously healthy, young adults
(1, 14). Of the extracellular GAS products, streptococcal
superantigens, including streptococcal pyrogenic exotoxin A (SPE-A) and
SPE-B, have been implicated in the pathogenesis of streptococcal TSS
and necrotizing fasciitis (1, 14). Properties of these
pyrogenic exotoxins include pyrogenicity, the ability to induce vast
lymphocyte proliferation, and the enhancement of host susceptibility to
endotoxin shock. Superantigens are microbial proteins capable of
activating a large proportion of lymphocytes, thereby causing an
excessive release of proinflammatory cytokines (5, 6).
We have previously demonstrated that patients with invasive GAS disease
are relatively deficient in antibodies against SPE-A and SPE-B compared
with healthy individuals (7, 8). In addition, anti-SPE-A
antibodies were associated with a favorable outcome of TSS
(8). In vivo and in vitro studies have suggested that intravenous immunoglobulin (IVIG) preparations would be beneficial as
adjunctive treatment for patients with severe GAS infections (10,
11). The mechanism by which IVIG may improve clinical outcome in
the setting of acute infectious diseases has not been totally
elucidated. Nevertheless, in vitro studies have revealed that IVIG has
neutralizing activity against a large variety of superantigens released
by clinical GAS isolates. Evidence has been presented that the
superantigen-counteracting effect of IVIG is conferred to patients
following treatment (10). However, sera that contain
exotoxin-specific antibodies do not always inhibit exotoxin-induced
proliferation (9). In addition, several studies have shown
variation between individuals and even between different IVIG
preparations with regard to the levels and neutralizing capacities of
SPE-specific antibodies (9, 10).
The present study was designed to investigate the average functional
capacities of polyclonal immunoglobulin G (IgG) antibodies to bind
SPE-A and SPE-B, comparing sera from patients with fatal GAS infections
with sera from healthy individuals and with IVIG preparations. The
affinity values obtained represent the overall binding properties
of a heterogeneous population of antigen-specific serum antibodies
as measured by competitive enzyme-linked immunosorbent assay
(ELISA) and are therefore described as relative avidities.
Sera.
In the period from 1994 to 1998, serum samples from six
Dutch patients (mean age, 52 years; age range, 30 to 74 years) who died
of streptococcal TSS and from eight healthy individuals (mean age, 41 years; age range, 25 to 61 years) were taken as sources of polyclonal
anti-SPE-A (
-SPE-A) and -SPE-B antibodies. Cases of TSS were
defined by previously published criteria (16). From one
patient, there was no more serum available for the estimation of the
relative avidity for SPE-B. Informed consent was obtained from the
patients or their relatives and from all controls. Six of the controls
were donors at a blood bank (Stichting Rode Kruis Bloedbank, Utrecht,
The Netherlands), and two were coworkers in our own department. We also
studied -SPE-A and -SPE-B antibodies in six batches of IVIG
preparations (Sandoglobulin; Sandoz, Basel, Switzerland) each dissolved
to a concentration of 3 mg/ml. Sera were stored at 
70°C until use.
Antigens.
SPE-A was purified from GAS strain NY-5 by
ultrafiltration, ethanol precipitation, and anion-exchange
chromatography as described by Mascini et al. (6). SPE-B,
isolated from GAS strain MGAS 1719 as described by Kapur et al.
(4), was kindly provided by J. M. Musser (Houston,
Tex.). SPE-A and SPE-B were more than 99% pure, as determined by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with
silver staining (6).
Detection of -SPE-A and -SPE-B antibodies.
-SPE-A and
-SPE-B antibody levels were determined by exotoxin-specific ELISAs
(6). Unless otherwise mentioned, all incubation steps were
performed at 37°C. Phosphate-buffered saline supplemented with 0.1%
Tween 20 was used in the washing procedures. ELISA plates (96-well,
flat bottom microtiter; Costar 2595, Cambridge, Mass.) were coated for
1 h with 100 µl of SPE-A or SPE-B (0.5 µg/ml of saline).
Blocking (1 h) was performed with 0.1% gelatin. Afterwards, the plates
were incubated with serum, serially diluted in washing buffer
(dilutions ranged from 1:30 to 1:10,000), for 1 h. The plates were
then subjected to incubation with peroxidase-labeled sheep
anti-human IgG antibodies (ICN Biochemicals, Amsterdam, The
Netherlands). A tetramethylbenzidine (2%
[wt/vol])-hydrogen peroxide (0.02% [wt/vol]) mixture
was used as chromogenic substrate. Optical densities in the wells were
determined with an ELISA microplate reader (Biorad, Hercules, Calif.)
operating at 450 nm. End-point titers at an optical density of 0.500 were calculated by linear regression and expressed as log dilution
factors. High-titer reference serum from one of the healthy individuals
was used as a positive control. The tested serum antibody levels were
expressed in arbitrary units (percentage of the titer of the
simultaneously tested positive reference serum) and geometrically
averaged as depicted in Fig. 1. IVIG
batches showed high titers for both -SPE-A and -SPE-B antibodies
compared with sera from patients and controls. No significant differences in antiexotoxin titers between patients and controls (Mann-Whitney test) were observed, although the patients showed a
larger proportion of low (<10 U/ml; log = 1) -SPE-A levels than controls (
2 = 5.091; P < 0.025).
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FIG. 1.
Individual titers of polyclonal IgG antibodies against
SPE-A (A) and SPE-B (B) in sera from patients with fatal GAS disease,
in sera from controls, and in IVIG batches. No significant differences
in antiexotoxin titers between patients and controls were observed,
although patients showed a larger proportion of low (<10 U/ml;
log = 1) -SPE-A levels than healthy individuals
(2 = 5.091; P < 0.05). AU,
arbitrary units.
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|
Relative avidity.
Relative avidities
(Kd's) of human serum antibodies for SPE-A and
SPE-B were estimated based on the following formulas and assumptions.
If the equation [Ag] + [Ab]
k2
k1 [AgAb], where
[Ag], [Ab], and [AgAb] represent the concentrations of antigen,
antibody, and antigen-antibody complexes, respectively, and
k1 and k2 represent the
association and dissociation velocities, respectively, is considered, then the avidity at equilibrium can be described by the following:
![K<SUB>d</SUB><UP> = </UP>[<UP>Ag</UP>]<UP> · </UP>[<UP>Ab</UP>]<UP>/</UP>[<UP>AgAb</UP>]<UP> = </UP>k<SUB><UP>2</UP></SUB><UP>/</UP>k<SUB><UP>1</UP></SUB><UP> = 1/</UP>K<SUB>a</SUB>](/content/vol6/issue6/fulltext/977/img001.gif)
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(1)
|
where
Ka is the association constant.
Subsequently, another parameter, the fraction of bound antibody
(
r), can be defined
by the equation
![r<UP> = </UP>[<UP>AgAb</UP>]<UP>/</UP>[<UP>Ab<SUB>tot</SUB></UP>]<UP> = </UP>[<UP>AgAb</UP>]<UP>/</UP>([<UP>AgAb</UP>]<UP> + </UP>[<UP>Ab</UP>])<UP> =</UP>](/content/vol6/issue6/fulltext/977/img002.gif)
|
(2)
|
where Ab
tot is the total amount of bound and free
antibodies. Substitution of equation 1 in equation 2 results in
![r<UP> = 1/</UP>(<UP>1 + </UP>K<SUB>d</SUB><UP>/</UP>[<UP>Ag</UP>])<UP> = </UP>[<UP>Ag</UP>]<UP>/</UP>([<UP>Ag</UP>]<UP> + </UP>K<SUB>d</SUB>)](/content/vol6/issue6/fulltext/977/img004.gif)
|
(3)
|
This means that when half of the antibody-binding sites are
occupied, then [Ag]/([Ag] +
Kd) = 0.5, which means that
Kd =
[Ag].
In practice,
Kd's of human serum IgG for SPE-A
and SPE-B were determined by using competitive ELISAs for SPE-A and
SPE-B.
Each exotoxin was serially diluted (10
4 to
10
7 g/liter) in uncoated, 96-well, flat-bottom microtiter
plates
(Greiner GmbH, Frickenhausen, Germany) and mixed 1:1 with a
fixed
concentration of the serum to be tested. The fixed serum
concentrations
we used in the avidity tests were deduced from the
ELISA-obtained
titers shown in Fig.
1: concentrations from the linear
part of
the ELISA curve corresponding to 80% of the maximum values
obtained
by ELISA were used. This means that different concentrations
were
used for individual serum samples or immunoglobulin preparations,
depending on the antibody titers of the samples to be tested.
The
exotoxin-antibody mixtures were incubated at 37°C for 1 h
(incubation times of up to 24 h longer yielded results similar
to
those obtained with the 1-h incubation). Separately, 96-well,
flat-bottom microtiter ELISA plates (Costar 3590, Cambridge, Mass.)
were coated with either SPE-A or SPE-B and blocked as described
above.
Aliquots (100 µl) of the exotoxin-antibody mixtures were
pipetted
into the coated plates and incubated for 1 h; the concentration
of
free antibody was then determined by competitive ELISA. A regular
ELISA
was performed with the same plate used for the competitive
ELISA in
order to establish the absorbance value corresponding
to half the
antibody concentration used in the competitive ELISA.
Next, the plates
were incubated with peroxidase-labeled sheep
anti-human IgG and the
ELISAs were developed as described above.
Optical densities in the
wells were determined with an ELISA microplate
reader (Bio-Rad)
operating at 450 nm. Since the antibody concentration
that yields half
saturation of the coated antigen corresponds
to the reciprocal of the
relative avidity, the
Kd values of the
antibodies tested for SPE-A or SPE-B were read from the curve
of the
competitive ELISA at the point where the concentration
of exotoxin
equaled the value found in the regular ELISA, as shown
in Fig.
2. Given a molecular mass of 28 kDa for
both exotoxins,
the molar inhibitory concentrations required for 50%
inhibition
could be determined. Since
Kd values
estimated in this way could
vary somewhat with the coating
concentrations used and polyclonal
antibodies may have a broad array of
Kd's, the term relative avidity
rather than
Kd value is used in this paper. Polyclonal
antibodies
derived from different sources, sera from patients with
fatal
streptococcal TSS, sera from healthy controls, and different
batches
of IVIG preparations, were tested.
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FIG. 2.
Principles of competitive ELISA used for the
determination of the relative avidity of polyclonal antibodies for
SPE-A. (A) Regular ELISA to determine the serum concentration (x) used
in the competitive ELISA and to establish the absorbance value
corresponding to half the antibody concentration (1/2x). (B)
Competitive ELISA. The relative avidity is deduced at the point where
the concentration of exotoxin equals the value obtained by regular
ELISA (K). OD450, optical density at 450 nm.
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|
There was a marked variation in the relative avidities estimated for
both exotoxins between individual antibody sources (Fig.
3). Values for SPE-A varied from 4 × 10
7 to 1 × 10
10 M and values for
SPE-B varied from 3 × 10
9 to 1 × 10
11 M. Compared with IVIG batches, sera from control
individuals
showed slightly high, but not significantly different,
values
for SPE-A (7 × 10
10 M and 1 × 10
9 M, respectively) and SPE-B (2 × 10
11 M and 4 × 10
11 M, respectively),
as determined by Mann-Whitney analysis. We
observed that relative
avidities of antibodies for SPE-B were
higher in sera from controls and
in IVIG preparations than in
sera from patients (
P < 0.01). Although a similar trend was observed
as to the avidities
for SPE-A, there were no significant differences
between results for
the separate serum sources. Table
1 shows
the comparison between geometric mean
Kd values
for SPE-A and
SPE-B.
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FIG. 3.
Individual avidity values of IgG antibodies for SPE-A
(A) and SPE-B (B) in sera from patients with fatal GAS disease, in sera
from controls, and in IVIG batches. Avidities of antibodies for SPE-A
and SPE-B were higher in sera from controls and in IVIG preparations
than in sera from patients (P = 0.112 and P = 0.014, respectively).
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TABLE 1.
Relative avidities of IgG antibodies in sera from
patients with fatal GAS disease, in sera from healthy controls, and
in IVIG preparations
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|
In this study, we addressed the question of whether the relative
avidities of sera from patients with serious GAS disease
could be
related to the clinical outcome of disease. Despite the
small number of
serum samples tested, we perceived that the average
affinities of
polyclonal antibodies for both exotoxins tended
to be substantially
lower in sera from patients than in sera from
controls; with regard to
SPE-B, significant differences between
patients and controls were
found. In fact, mean relative avidities
for exotoxins A and B were
about 10-fold lower in the sera of
patients than in sera of controls,
which implies that about 10
times as many antibodies are required to
obtain a similar binding
of exotoxin in patient
sera.
Our data are in line with those of others who found an association, in
Scandinavian patients, between low capacities of sera
to neutralize
exotoxin-induced T-cell proliferation and serious
GAS infections
(
9). Interestingly, those authors reported that
the capacity
of human SPE-A- and SPE-B-specific antibodies to
neutralize the
mitogenic activity of the corresponding proteins
was highly variable.
We also noted a considerable variation in
relative avidity values, not
only within the groups of patients
and controls but also between
different IVIG batches. Remarkably,
the range of average affinity
values for SPE-B within control
sera and immunoglobulin preparations
appeared relatively small,
ranging from 1 × 10
11 to
5 × 10
11 M. Otherwise, the relative avidities of
-SPE-A antibodies within
these two groups showed an impressive
variation, with values between
3 × 10
8 M and 3 × 10
10 M. The fact that sera from controls showed
slightly higher relative
avidity values than immunoglobulin
preparations might be explained
by the procedures involved in the
manufacturing of immunoglobulin
preparations. The fairly high relative
avidity values presented
here are in line with a similar value
(1.8 × 10
10 M) for a mouse monoclonal IgG2a
antibody against SPE-A (
15).
The relative avidities of antibodies for SPE-B were significantly
higher than those for SPE-A in all three groups. In line
with this,
neutralization of SPE-A activity has been reported
to require serum
concentrations higher than those required by
other streptococcal
superantigens (
10). The antiexotoxin titers
as well as the
capacity of the antibodies to bind SPE-A and SPE-B
present in IVIG
preparations are likely to determine the neutralizing
activity against
streptococcal products and, thereby, whether
or not a particular batch
is beneficial for patients with severe
GAS disease. These data stress
the importance of screening IVIG
pools prior to use in the clinical
setting.
We developed a solid-phase assay with immobilized antigen to determine
the concentration of the free (not associated with
exotoxin) antibody.
We first incubated the antibodies and the
antigen in homogeneous
solution long enough to reach equilibrium,
and only then was the
concentration of free antibodies estimated
by ELISA. Indirect
competitive ELISA methods offer many advantages
as long as they are
used under conditions in which equilibrium
is reached and not
subsequently perturbed by the immunoassay (
2,
3). First,
only one of the components (antigen or antibody)
needs to be purified
(
2). Second, problems concerning steric
hindrance, antibody
valence, and antigen density and mobility
on the surface, which affect
the position of equilibrium on a
surface, are not encountered
(
2). Third, we are dealing with
unmodified molecules, since
no labeling of either the antigen
or the antibody is required. In
support of our method, a significant
correlation between competitive
enzyme immunoassays and methods
established by others (as long as
avidity values were read from
the linear part of the saturation curve
obtained at high saturation
of the antibody by the antigen) has been
reported (
2,
3,
13). One disadvantage of our approach is
that it may be difficult
to estimate relative avidities for sera with
low antibody levels.
Moreover, in the case of high-avidity values
requiring low concentrations
of exotoxin in the incubation mixture, a
shift of the equilibrium
in the fluid phase may be induced by the
relatively high SPE concentration
in the coating, thus yielding an
underestimation of the avidity
values (
3). Otherwise,
problems with competitive ELISA are
related to the restricted
sensitivity of the solid-phase assay;
the limiting affinity level of
antibodies in most test systems
appears to be 10
12 M, but
obviously the majority of antibodies in enzyme immunoassays
have lower
avidity constants (
12). Furthermore, we cannot provide
information as to the heterogeneity of antibody affinities in
a
polyclonal response, since this was beyond our goal of comparing
operational
affinities.
In conclusion, the relative avidities of antibodies for both toxins
were considerably lower in sera from patients with fatal
TSS than in
sera from controls or in IVIG batches, in addition
to the levels of
antibody being lower in the serum. It might be
argued that patients who
develop severe GAS disease have a deficiency
in high-avidity antibodies
against SPE-A and SPE-B, which is probably
best described by the
product of titer and relative avidity. The
variation between different
sera and immunoglobulin preparations
in SPE-binding capacity is likely
to reflect the variation in
SPE neutralization of these
samples.
| |
ACKNOWLEDGMENTS |
We thank J. M. Musser (Baylor College of Medicine, Houston,
Tex.) for supplying SPE-B.
This work was supported by the Dutch Praeventie Fonds, project no. 002824180.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Eijkman-Winkler
Institute for Microbiology, Infectious Diseases, and Inflammation, Utrecht University Hospital G04.614, P.O. Box 85500, NL-3508 GA Utrecht, The Netherlands. Phone: 31-302 507627. Fax: 31-302 541770. E-mail: e.m.mascini{at}lab.azu.nl.
| |
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Clinical and Diagnostic Laboratory Immunology, November 1999, p. 977-980, Vol. 6, No. 6
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Text
![1/(1 + [<UP>Ab</UP>]<UP>/</UP>[<UP>AgAb</UP>])](/content/vol6/issue6/fulltext/977/img003.gif)
