Exposure of IgG to an acidic environment results in
molecular modifications and in enhanced protective
activity in sepsis
Iglika K. Djoumerska-Alexieva
1,
*, Jordan D. Dimitrov
1,2,3,4,
*, Elisaveta N. Voynova
1
,
Sebastien Lacroix-Desmazes
2,3,4
, Srinivas V. Kaveri
2,3,4
and Tchavdar L. Vassilev
1
1 Department of Immunology, Stefan Angelov Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria
2 Centre de Recherche des Cordeliers, Universite
´
Pierre et Marie Curie Paris 6, France
3 Universite
´
Paris Descartes, France
4 INSERM U 872, Eq. 16, Paris, France
Introduction
The ability of antibodies to interact with one single or
with multiple structurally unrelated antigens (monore-
activity versus polyreactivity) is believed to be an
inherent property of each individual immunoglobulin
molecule. However, it has been previously shown by
us, as well as by others, that the in vitro exposure of

to engage in F(ab¢)
2
⁄ F(ab¢)
2
(idiotype ⁄ anti-idiotype) interactions and an
increased functional antigen-binding affinity are reported here. The newly
acquired ‘induced polyreactivity’ of low-pH buffer-exposed IgG is related
to structural changes in the immunoglobulin molecules, and is at least
partly attributable to the enhanced role of the hydrophobic effect in their
interactions with antigen. Our results suggest that data from many previous
studies on monoclonal and polyclonal IgG antibodies purified by low-pH
buffer elution from protein A or protein G immunoaffinity columns should
be reconsidered, as the procedure itself may have dramatically affected
their antigen-binding behavior and biological activity. Low-pH buffer-trea-
ted pooled therapeutic immunoglobulins acquire novel beneficial properties,
as passive immunotherapy with the pH 4.0 buffer-exposed, but not with
the native therapeutic intravenous immunoglobulin preparation, improves
the survival of mice with bacterial lipopolysaccharide-induced septic shock.
Abbreviations
ANS, 8-anilinonaphthalene-1-sulfonate; CRP, C-reactive protein; IFN-c, interferon-c; IVIg, intravenous immunoglobulin;
LPS, lipopolysaccharide; RU, relative units.
FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS 3039
A comparative study of seven licensed commercially
available pooled therapeutic intravenous immunoglob-
ulins (IVIgs) revealed that those produced using a frac-
tionation step at low pH were significantly more
polyreactive when tested on a complex mix of self-anti-
gens [8]. The molecular mechanisms responsible for the
effect of low-pH buffer exposure on IgG molecule
have remained, however, poorly understood. IVIg

IVIg acquired enhanced autoreactivity [8]. The first
aim of the study was to find out whether the same
broadening of IgG polyreactivity occurred when for-
eign antigens were used. The exposure of pooled
human IgG to a pH 4 buffer resulted in an increase
in its pre-existing binding to antigens present in an
Escherichia coli lysate, and in the appearance of some
new bands showing the acquisition of new antibody
reactivities (Fig. 1A). Interestingly, the same treatment
did not significantly change the reactivity to Bacillus
anthracis antigens. In contrast, the transient exposure
of IVIg to pH 2.8 buffer resulted in a significant
(P < 0.05) enhancement of immunoreactivity, and in
A
B
C
E
D
Fig. 1. Exposure of IgG to a pH 4 buffer results in increased antigen-binding polyreactivity. (A) Densitometric profiles of the reactivity of the
native (solid line) and low-pH buffer-exposed (dashed line) IVIg to Escherichia coli antigens. Migration distances (x-axis) expressed in pixels
were plotted against the intensity of binding (y-axis) expressed in relative units (RU) for each IVIg preparation. (B) Reactivity of native (lines 1
and 4), pH 4 buffer-exposed (lines 2 and 5) and pH 2.8 buffer-exposed (lines 3 and 6) IVIg with Bacillus anthracis antigens. The membranes
were incubated with two concentrations of IVIg: 100 lgÆmL
)1
(lines 1–3) and 50 lgÆmL
)1
(lines 4–6). (C) Increased binding of pH 4 buffer-
exposed IVIg to recombinant human IFN-c. (D) Increased binding of low-pH buffer-exposed IVIg to human factor H. In both panels, binding
of the native IVIg is indicated by a solid line, and that of the low-pH buffer-exposed IVIg is indicated by a dashed line. (E) Binding of two
commercially available IVIg preparations [Endobulin (solid line); Octagam (dashed line)] to human factor H. Data represent mean absorbance

gam). As expected, the binding of the second to fac-
tor H was significantly (P < 0.05) higher (Fig. 1E).
The same was true for all other antigens in the panel
(not shown).
Low-pH buffer exposure increases the
anti-idiotypic reactivity of IgG
After a pH 4 buffer exposure, the studied IVIg prepa-
ration showed enhanced binding to IVIg F(ab¢)
2
frag-
ments (Fig. 2A) as well as to autologous pooled IgM
molecules (Fig. 2B). In contrast, no increase in reactiv-
ity of the modified IVIg to Fc-c (Fig. 2C) or Fc-l
fragments (Fig. 2D) was observed.
Antigen-binding kinetics of low-pH
buffer-exposed IVIg
In order to obtain quantitative information on the
effect of low-pH buffer exposure on the reactivity of
IgG, we used real-time kinetic measurements of the
interaction of IVIg with C-reactive protein (CRP). The
binding profiles obtained after single injections of
native and of low-pH buffer-exposed IVIg were com-
pared. As shown in Fig. 3A, transient (5 min) exposure
of IVIg to pH 2.8 buffer resulted in an increase in the
reactivity towards human CRP, as detected by this
nonequilibrium binding assay. The reactivity of the
pH 4 buffer-exposed preparation was also elevated,
but to a much lower extent (approximately nine-fold).
The native IVIg preparation showed no detectable
binding to CRP at the concentration and during the

)3
) ± (2.0 · 10
)5
s
)1
). The equilibrium dissociation
constant for CRP and low-pH buffer-exposed IVIg
was 69.1 lm. The absence of detectable binding or
negligible responses precluded the estimation of reli-
able values of kinetic parameters for the native and
the pH 4 buffer-exposed IVIg preparations.
Enhanced role of hydrophobicity in the binding
of a pH 4.0 buffer-treated monoclonal IgG
antibody to its target antigen
To evaluate the types of intermolecular interactions in
antigen binding, pH and salt concentration screening
assays were performed. This study was carried out
using the mouse monoclonal Z2 antibody, which
behaves in its native form as a typical monoreactive
antibody, as it interacts only with mouse IgG
2a
[14].
The interaction of the native Z2 antibody with its
immobilized target antigen was highly pH-dependent,
and characterized by a bell-shaped curve with a bind-
ing optimum at neutral pH (Fig. 4A). On the other
hand, the interaction of low-pH buffer-exposed Z2
antibody was much less dependent on the pH of the
buffer. From pH 4.5 upwards, the binding reached a
plateau and became almost independent of further

two independent experiments are shown.
Molecular modifications in low pH-exposed IgG I. K. Djoumerska-Alexieva et al.
3042 FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS
fluorescence properties with the polarity of the envi-
ronment. Thus, the transition from a polar to a non-
polar (hydrophobic) environment results in a dramatic
increase in its fluorescence signal. This property and
the ability of ANS to bind to proteins make it a widely
used molecular probe for the evaluation of hydropho-
bicity of proteins, as well as for exploring structural
alternations in protein molecules [15–18].
Incubation of native IVIg in the presence of ANS
resulted in a modest change in the fluorescence signal
(Fig. 4C). Similar spectral characteristics were mea-
sured in the case of IVIg exposed to a pH 4 buffer,
implying the absence of a significant increase in the
total hydrophobicity of the immunoglobulins. The lack
of correlation between these findings and the data
shown in Fig. 4A,B could well be explained by the dif-
ferent IgG preparations studied (pooled, polyclonal
versus monoclonal) and the different sensitivities of the
methods used. Dramatic changes in the fluorescence
characteristics of ANS were seen in the presence of
pH 2.8 buffer-exposed IVIg. Thus, a considerable
increase in the fluorescence intensity and a blue shift in
the fluorescence maxima were observed. These effects
were observed at different concentrations of ANS
(Fig. 4D). These findings further confirmed the results
from the pH and ionic strength dependencies of the
interactions, demonstrating the increased hydrophobic-

I. K. Djoumerska-Alexieva et al. Molecular modifications in low pH-exposed IgG
FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS 3043
molecules to an acidic milieu would result in structural
modifications. Indeed, an increase in the fluorescence
intensity of the pooled IgG preparation and a slight
red shift in the emission maxima following its exposure
to a pH 2.8 buffer were detected by fluorescence spec-
troscopy (Fig. 5). This effect is consistent with a
change in the positions of tryptophan(s) in the immu-
noglobulins. The red shift in the emission maxima is
indicative of the relocation of tryptophan(s) to a more
polar environment. In contrast, treatment of the same
preparation with a pH 4 buffer did not change the
tryptophan fluorescence of the same molecules.
Increase in the relative functional antigen-binding
affinity of a monoclonal antibody after its
exposure to a pH 4 buffer
The relative functional affinities of the native and the
low-pH buffer-exposed monoclonal Z2 antibody were
analyzed by thiocyanate elution ELISA [20]. In the
elution assay, 0–3.0 m potassium thiocyanate was used
to disrupt the binding of the Z2 antibody to its target,
mouse IgG
2a
. The functional affinity was defined by
the molar concentration of potassium thiocyanate
required for a 50% reduction in binding as detected in
ELISA at A
405 nm
. The modified mouse monoclonal

Discussion
The brief exposure of polyclonal and at least some
monoclonal IgG preparations to a low-pH buffer
results in an alteration of the immunoglobulin struc-
ture, and in the acquisition of enhanced antigen recog-
nition behavior and new biological activities. Our data
show that the changes fall short of denaturation of the
immunoglobulin molecules. The main argument in
support of this claim that low-pH-modified IgG
molecules fully retain their F(ab¢)
2
-dependent and
Fig. 5. Increase in the fluorescence intensity of pooled IgG after
low-pH buffer exposure. Spectrofluorometric analyses of native
(solid line), pH 4 buffer-exposed (dotted line) and pH 2.8 buffer-
exposed (dashed line) IVIg. The ordinate represents fluorescence
intensity in RU.
Fig. 6. Increased functional affinity of a mouse monoclonal Z2 anti-
body after its exposure to a pH 4 buffer. Thiocyanate elution ELISA
was performed as described in Experimental procedures. The func-
tional affinity is represented by the molar concentration of potas-
sium thiocyanate required for a 50% reduction in binding as
detected at A
405 nm
. The results represent the average of at least
three independent measurements, with the standard deviation indi-
cated by error bars (gray bar, native Z2 antibody; black bar, pH 4
buffer-exposed Z2 antibody).
Molecular modifications in low pH-exposed IgG I. K. Djoumerska-Alexieva et al.
3044 FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS

and affects the IgG molecules themselves.
A possible explanation for the finding of new anti-
gen-binding specificities after exposure to low-pH con-
ditions is the induction of structural rearrangements in
the variable region of the antibody. Indeed, we
observed changes in the tryptophan fluorescence char-
acteristics of IVIg after its exposure to a low-pH (2.8)
buffer. The increase in the fluorescence intensity and
the red shift in the emission maximum are signs of a
change in the position of tryptophan(s) in the IgG
molecules – a mark of the existence of a structural
modification in the polypeptide chains of the immuno-
globulins. In addition, our results revealed that the
interactions of low-pH buffer-exposed IgG are less
dependent on changes in the ionic strength or the pH
of the medium. Such binding behavior is typical of
protein–protein bonds that rely on nonpolar types of
interaction (hydrophobic effect and van der Waals
contacts). Indeed, the increase in the hydrophobicity
may well be explained by exposure of previously bur-
ied hydrophobic amino acids to the solvent, as shown
previously for IgG treated with chaotropic agents [23].
By using the fluorescence molecular probe for hydro-
phobicity of proteins (ANS), we confirmed that the
exposure of IgG to a low-pH buffer results in molecu-
lar modifications characterized by a considerable
increase in their surface-exposed hydrophobicity. The
antigen-binding behavior of low-pH buffer-exposed
IgG preparations was enhanced for some, but not all,
antigens tested (e.g. IgG Fc fragments). We are cur-

(pH 7.4) alone (gray lines). The protective activity of the modified
IVIg is retained, even when its administration has been postponed
for 1 h (E). *P < 0.05, Mann–Whitney test.
I. K. Djoumerska-Alexieva et al. Molecular modifications in low pH-exposed IgG
FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS 3045
plasticity of their paratopes. Urea and low-pH buffer
exposure are both known to induce some degree of
melting of the protein conformation. The increased poly-
reactivity observed could well be explained by limited
melting of immunoglobulin molecules by either of
these agents.
Our previous data have indicated that treatment of
some IgG antibodies with different redox-active agents
is also able to enhance antigen-binding polyreactivity
by modulating the properties of the antigen-combining
sites of some, but not all, studied IgG antibodies. The
binding behavior of some, generated in response to
repeated immunizations and expected to have rigid,
high-affinity binding sites, is not modified by exposure
to these conditions [5]. The precise mechanisms that
make an individual IgG molecule resistant to polyreac-
tivity-inducing treatment remain to be determined.
Redox-active agents (heme, iron ions, reactive oxygen
species) are released in vivo in inflammation sites as
well as after trauma, hemorrhages, etc. Exposure to
low pH is part of the production process for some
commercial IVIg preparations, and is used on a daily
basis for IgG immunoaffinity purification.
The dramatically increased antigen-binding polyreac-
tivity of polyclonal and of some monoclonal IgG anti-

B). The low-pH buffer elution of the same fraction
may also enhance its capacity to engage in
F(ab¢)
2
⁄ F(ab¢)
2
(idiotype ⁄ anti-idiotype) interactions
with other immunoglobulin molecules (see Fig. 2).
IVIg preparations are used in patients with primary
and secondary immunodeficiencies, as well as in an
increasing number of autoimmune and inflammatory
diseases. To the best of our knowledge, no compara-
tive clinical studies on the immunomodulatory effects
of IVIg preparations produced by different protein
fractionation technologies have been performed so far.
Data from this and from an earlier study [8] strongly
suggest that licensed therapeutic IVIgs exposed to pro-
duction steps at low pH do acquire new, clinically rele-
vant, properties. Their use could be beneficial in the
early stages of sepsis, which are characterized by
uncontrolled production of proinflammatory mediators
(‘cytokine storm’). Previous studies have shown that
binding to bacterial LPS is not affected in IgG mole-
cules modified by protein-destabilizing agents [5],
strongly suggesting that the prevention of LPS-induced
sepsis death is due to the ability of this preparation
to attenuate the hyperreactivity of body defense
mechanisms.
In addition to sepsis, there is an increasing number
of emerging infectious diseases in which the severe gen-

preparation, were prepared as described previously [32,33].
Pure human Fc-l fragments, obtained from a patient with
l-heavy chain disease, were a gift from L. Mouthon (Cochin
Hospital, Paris).
The Z2 antibody and IVIg samples were diluted in 0.1 m
sodium acetate buffer (pH 4.0 or 2.8) and incubated for
5 min. The pH was then brought to 7.0, and the samples
were dialyzed against NaCl ⁄ P
i
(pH 7.2) [8]. The pH 4.0
buffer was chosen because several commercial IVIg prepa-
rations are produced using a fractionation step with this
pH value. Buffers of pH 2.8 are widely used for the isola-
tion of pure IgG by affinity chromatography.
Immunoblot analysis
The total lysate from a nonpathogenic strain of B. anthra-
cis was kindly provided by S. Mesnage (Centre de Recher-
che des Cordeliers, Paris, France). A lysate of E. coli was
prepared as described elsewhere [5]. Both bacterial antigen
extracts were subjected to 10% SDS ⁄ PAGE and trans-
ferred to nitrocellulose membranes (Scheicher & Schuell,
Dassel, Germany) with a Mini Transfer Blot system (Bio-
Rad, Richmond, CA, USA) in a buffer containing 48 mm
Tris, 110 mm glycine, and 20% (v ⁄ v) methanol. Then, they
were incubated for 1 h at room temperature in NaCl ⁄ Tris
containing 0.3% Tween-20. Membranes were further cut
into strips or fixed in a miniblot system, and incubated
for 1 h at room temperature with the native, the pH 4.0
buffer-exposed or the pH 2.8 buffer-exposed IVIg prepara-
tions (at 0.1 mgÆmL

)1
human C3 (both
from Calbiochem), 10 lgÆmL
)1
human factor H,
10 lgÆmL
)1
human factor B (both from Complement
Technology, TX, USA), 20 lgÆmL
)1
porcine thyroglobulin,
20 lgÆmL
)1
rabbit tubulin, and 10 lgÆmL
)1
bovine myelin
basic protein (all three from Sigma-Aldrich), for 2 h at
room temperature. Plates were blocked with 0.25–
0.4% (v ⁄ v) Tween-20 in NaCl ⁄ P
i
for 2 h. After washing
with NaCl ⁄ P
i
containing 0.05% Tween-20, the plates were
incubated overnight at 4 °C (in the case of IFN-c) or for
2 h at 25 °C (in the case of other proteins) with increasing
concentrations of the immunoglobulin preparations under
study. The plates were then extensively washed, and goat
anti-(human IgG) (c-chain-specific) coupled to alkaline
phosphatase was added and incubated for 1 h at room tem-

3
, 0.05 m Na
2
CO
3
, 0.14 m
NaCl, 0.05% (v ⁄ v) Tween-20] with a pH in the range
8.5–12. In the ELISA with increasing salt concentrations,
the Z2 antibody was diluted to 15 lgÆmL
)1
in buffers with
0–4 m sodium chloride. After a 2 h incubation step under
the described conditions, the plates were washed and
further incubated with an alkaline phosphatase-conjugated
goat anti-mouse IgG
2b
(PharMingen, San Diego, CA,
USA) for 1 h at room temperature. The following steps of
the assay were performed as described above. The results
were represented in RU. The binding at pH 7.0 or in the
presence of 0 m NaCl buffers, respectively, was referred to
as 1 RU.
The abilities of both IVIg variants to engage in idio-
type ⁄ anti-idiotype interactions were compared by ELISA.
Polystyrene plates were coated with F(ab¢)
2
or Fc IVIg
fragments, with pooled IgM, or with pure Fc-l fragments
(all at 10 lgÆmL
)1

5–0.039 lm at a flow rate of 10 lLÆmin
)1
. The association
and dissociation phases of the interaction were monitored
for 5 min. The regeneration of the chip surface was
performed using a 5 m solution of guanidine-HCl (Sigma-
Aldrich). The binding to the surface of the uncoupled
control flow cell was always subtracted from the binding to
the protein-coated flow cells. biaevaluation software (ver-
sion 4.1; Biacore) was used for the calculation of the kinetic
rate constants. Calculations were performed by global
analysis of the experimental data using the kinetic models
included in the software, fitting the data with lowest value
of v
2
.
Fluorescence spectroscopy
Intrinsic emission spectra measurements of the native and
of the low-pH buffer-exposed IVIg were performed on a
Hitachi F-2500 spectrofluorometer. Samples of the IVIg
were exposed at 4 °C to pH 4 or pH 2.8 acetate buffers.
After incubation, the samples were dialyzed against
NaCl ⁄ P
i
. All analyses were carried out at 25 °C, using 1 cm
quartz cuvette. The samples were diluted in NaCl ⁄ P
i
to a
final concentration of 2 lm. A wavelength of 295 nm,
which excites tryptophans, was used for the fluorescence

sured using a goat anti-mouse IgG
2b
as described above.
Experimental septic shock
Outbred ICR mice were purchased from the Breeding Farm
of the Bulgarian Academy of Sciences. The experimental
protocols were approved by the Animal Care Commission
of the Institute of Microbiology, in accordance with
National and European Regulations. The number of ani-
mals used was kept at the minimum that still ensured statis-
tical significance of survival differences between the
experimental groups. Septic shock was induced in 16–18-
week-old animals by the intraperitoneal administration of
400 lg of bacterial LPS (from E. coli B 055:B5, Sigma-
Aldrich, #L2880). Minutes later, groups of mice (15 per
group) were injected intravenously with increasing doses of
the native IVIg or of the low-pH buffer-exposed IVIg prep-
aration, or with NaCl ⁄ P
i
alone. Survival was observed for
5 days. In a separate experiment, the native and the modi-
fied IVIg (500 mgÆkg
)1
, 10 animals per group) were infused
1 h after the administration of LPS. Any effect of a treat-
ment started at this time-point should be the result of its
influence on the pathophysiological mechanisms of the sep-
sis syndrome [34].
Statistical analysis
Statistical analyses were performed using graphpad prism,

IgG in intravenous immunoglobulin preparations.
Proteomics 9, 2253–2262.
3 Dimitrov J, Roumenina L, Doltchinkova V, Mihaylova
N, Lacroix-Desmazes S, Kaveri S & Vassilev T (2007)
Antibodies use heme as a cofactor to extend their path-
ogen elimination activity and to acquire new effector
functions. J Biol Chem 282, 26696–26706.
4 Dimitrov J, Roumenina L, Doltchinkova V & Vassilev
T (2007) Iron ions and haeme modulate the binding
properties of complement subcomponent C1q and of
immunoglobulins. Scand J Immunol 65, 230–239.
5 Dimitrov JD, Ivanovska ND, Lacroix-Desmazes S,
Doltchinkova VR, Kaveri SV & Vassilev TL (2006)
Ferrous ions and reactive oxygen species increase
antigen-binding and anti-inflammatory activities of
immunoglobulin G. J Biol Chem 281, 439–446.
6 McIntyre J, Wagenknecht D & Faulk W (2005)
Autoantibodies unmasked by redox reactions.
J Autoimmun 24, 311–317.
7 McMahon MJ & O’Kennedy R (2000) Polyreactivity as
an acquired artefact, rather than a physiologic property
of antibodies: evidence that monoreactive antibodies
may gain the ability to bind to multiple antigens after
exposure to low pH. J Immunol Methods 241, 1–10.
8 Djoumerska I, Tchorbanov A, Pashov A & Vassilev T
(2005) The autoreactivity of therapeutic intravenous
immunoglobulin (IVIG) preparations depends on the
fractionation methods used. Scand J Immunol 61,
357–363.
9 Notkins A (2004) Polyreactivity of antibody molecules.

(1999) 1-Anilino-8-naphthalene sulfonate as a protein
conformational tightening agent. Biopolymers 49,
451–458.
18 Sudhakar K & Fay P (1996) Exposed hydrophobic sites
in factor VIII and isolated subunits. J Biol Chem 271,
23015–23021.
19 Harding SE & Chowdhry BZ (2001) Protein–Ligand
Interactions: Structure and Spectroscopy. Oxford
University Press, Oxford.
20 Pullen G, Fitzgerald M & Hosking C (1986) Antibody
avidity determination by ELISA using thiocyanate
elution. J Immunol Methods 86, 83–87.
21 Coutinho A & Avrameas S (1992) Speculations on
immunosomatics: potential diagnostic and therapeutic
value of imune homeostasis concepts. Scand J Immunol
36, 527–532.
22 Wymann S, Ghielmetti M, Schaub A, Baumann M,
Stadler B, Bolli R & Miescher S (2008) Monomerization
of dimeric IgG of intravenous immunoglobulin (IVIg)
increases the antibody reactivity against intracellular
antigens. Mol Immunol 45, 2621–2628.
23 Dimitrov J, Lacroix-Desmazes S, Kaveri S & Vassilev T
(2007) Transition towards antigen-binding promiscuity
of a monospecific antibody. Mol Immunol 44, 1854–1863.
24 Vassilev T, Gelin C, Kaveri SV, Zilber MT, Boumsell L
& Kazatchkine MD (1993) Antibodies to the CD5 mol-
ecule in normal human immunoglobulins for therapeu-
tic use (intravenous immunoglobulins, IVIg). Clin Exp
Immunol 92, 369–372.
25 Vassilev TL, Kazatchkine MD, Van Huen JPD,

virus spread within and between hosts. Am J Pathol
168, 176–183.
31 Venter M, Burt F, Blumberg L, Fickl H, Paweska J &
Swanepoel R (2009) Cytokine induction after labora-
tory-acquired West Nile virus infection. N Engl J Med
360, 1260–1262.
32 Debre M, Bonnet MC, Fridman WH, Carosella E,
Philippe N, Reinert P, Vilmer E, Caplan C, Teillaud JL
& Griscelli C (1993) Infusion of Fcg fragments for the
treatment of children with acute immune thrombocyto-
penic purpura. Lancet 342, 945–949.
33 Hurez V, Kazatchkine MD, Vassilev T, Ramanathan S,
Basuyaux B, Pashov A, De-Kozak Y, Bellon B &
Kaveri SV (1997) Pooled normal human polyspecific
IgM contains neutralizing antiidiotypes to IgG
autoantibdies of autoimmune patients and protects
from autoimmune disease. Blood 90, 4004–4013.
34 Cohen J (2002) The immunopathogenesis of sepsis.
Nature 420, 885–891.
Molecular modifications in low pH-exposed IgG I. K. Djoumerska-Alexieva et al.
3050 FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS