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*
Division of Allergy and Immunology, University of Pennsylvania School of Medicine and
Laboratory of Pathology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Given its ability to react with a large amount of Ig molecules, a B
cell SAg could inflict tissue damage through a number of inflammatory
mechanisms. Its interaction with cytophilic IgG, IgE, or IgA molecules
could lead to the cross-linking of the respective Ig FcRs on
inflammatory cells, thereby resulting in the release of inflammatory
mediators. Indeed, SpA, protein L, and protein Fv induce histamine
release from human basophils by interacting with the Fab region of IgE
molecules that are bound to Fc
R on these cells (23, 24, 25); protein L
and protein Fv also degranulate human mast cells (24, 25). The
interaction of a B cell SAg with fluid-phase IgG could lead to immune
complex-mediated tissue injury, a possibility that has not been
formally investigated. However, based on our recent findings (26), it
is now known that the immune complexes that are formed by a B cell SAg
with reactive serum Igs cause activation of the classical complement
cascade.
For many years, investigators have exploited the Arthus reaction as a model of in vivo immune complex-mediated tissue injury (27, 28, 29, 30). In the classical model of the Arthus reaction, animals are immunized with an Ag until they have appreciable levels of precipitating IgG Abs. Intradermal (i.d.) injection of the same Ag elicits a local inflammatory response (28). This response is characterized grossly by erythema, edema, and hemorrhage and microscopically by a prominent polymorphonuclear cell (PMN) infiltrate that peaks at 8 h after cutaneous challenge.
In the present study, we sought to determine whether a B cell SAg could elicit immune complex-mediated tissue injury. We now show that rabbits injected i.d. with the model B cell SAg, SpA, do not develop a cutaneous Arthus reaction, as previously reported (30). However, when rabbits are pretreated i.v. with human IgG (hIgG) from healthy donors, they do develop a cutaneous reaction with the histologic features of the Arthus reaction at the sites that were injected i.d. with SpA. This reaction is mediated by the Fab-binding site on SpA, since it was not induced in animals that were pretreated with (hIgG) depleted of VH3+ molecules (VH3-hIgG). These data provide the first evidence that the interaction of a B cell SAg, SpA, with its reactive (VH3+) Igs leads to an immune complex-mediated inflammatory reaction in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male and female New Zealand white rabbits (2–4 kg) were obtained from Ace Animals (Boyertown, PA) and housed in the animal facility at the University of Pennsylvania (Philadelphia, PA).
Administered reagents
Lyophilized, pooled normal hIgG (kindly provided by Sandoz Pharmaceutical, East Hanover, NJ) was reconstituted to 60 mg/ml with sterile 0.9% saline. rSpA (Repligen, Needham, MA) and human serum albumin (HSA) (low endotoxin) (Calbiochem, La Jolla, CA) were sterile filtered through 0.2-µm filters (Millipore, Bedford, MA).
Preparation of hyperiodinated SpA (Mod SpA)
rSpA was hyperiodinated to abrogate its IgG Fc-binding activity, as previously described (6).
Conjugation of Mod SpA to Sepharose 4B
We suspended 300 µg of freeze-dried CNBr-activated Sepharose 4B (Seph) (Sigma, St. Louis, MO) in 12.5 ml of 1 mM HCl; the Seph was subsequently rotated for 15 min at room temperature to swell the beads (300 µg of Seph yields 1 ml of gel.) The beads were then centrifuged at 2500 rpm for 2 min at room temperature. The supernatant was aspirated, and the beads were resuspended in 10 ml of 1 mM HCl and centrifuged as described above. Following the repetition of these steps, the beads were washed in coupling buffer (0.1 M NaHCO3 and 0.5 M NaCl (pH 8.3)). The supernatant was aspirated, and 4 mg Mod SpA plus coupling buffer or coupling buffer alone (for a total volume of 1.5 ml) was added to 1 ml of swelled Seph. Following rotation for 2 h, the beads were centrifuged as described above and washed once in coupling buffer. Next, 2 ml of 0.2 M glycine was added to 1 ml of swelled Seph and rotated for 1 h at room temperature. The Seph beads were centrifuged, washed with an acetate buffer (0.1 M sodium acetate and 0.5 M NaCl (pH 4.0)), and then washed with a Tris buffer (0.1 M Tris-HCl and 0.5 M NaCl (pH 8.0)). This procedure was repeated two more times. The Mod SpA-conjugated and unconjugated Seph beads were stored in 0.1% BSA/PBS with 0.02% sodium azide at 4°C until needed. At the time of use, the Seph beads were washed extensively with the appropriate buffer.
Depletion of VH3+ IgG from pooled hIgG
The pooled normal hIgG was passed over either a Mod SpA-conjugated Sepharose column (Mod SpA-Seph column) or an unconjugated Sepharose column (Seph column). Polyprep chromatography columns (Bio-Rad, Hercules, CA) were packed with 10 ml of either Mod SpA-Seph or Seph alone and washed with 100 ml of 1x running buffer (1:5 dilution of 5x running buffer (pH 7.0): 0.079 M KH2PO4, 0.48 M Na2HPO4, and 0.77 M NaCl). Next, 1 ml of pooled IgG (60 mg/ml) diluted with 250 µl of 5x running buffer was added to each column and allowed to pass into the Seph. The column was then closed, and the IgG was incubated in the column for 5 min. This procedure was repeated twice for each column. Afterward, 10 ml of 1x running buffer was added, and 1 ml fractions were collected. This process was repeated until the A280 of the fractions was <0.100. The column was subsequently flushed with 50 ml of 1x running buffer. The IgG concentrations of the collected fractions were determined by the following method: A280/extinction coefficient for IgG (1.43). Effluent fractions with a concentration of >1 mg/ml were pooled together and dialyzed two times against PBS at 4°C. The samples were concentrated in an Ultrafree-50 centrifugal filter (Millipore), and IgG concentrations were determined again as described above. The effluent fractions were then passed over their respective columns a second time, and the dialysis and concentration procedures were repeated.
ELISA for determining the binding of IgG fractions to Mod SpA or SpA
Half-area microtiter plate wells (Costar, Cambridge, MA) were coated with 100 µl Mod SpA, SpA, or BSA (Calbiochem) at 10 µg/ml in PBS overnight at 4°C. Each well was subsequently saturated with 100 µl 1% BSA/PBS for 2 h at room temperature. The wells were washed three times with 0.05% Tween-20 (Sigma) in PBS. We incubated 100 µl aliquots of varying concentrations of unfractionated hIgG or hIgG fractions from the Mod SpA-Seph column or the Seph column for 2 h at room temperature. The wells were washed as described above, and peroxidase-conjugated goat F(ab')2 anti-hIgG Fc Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) was added for 1 h at room temperature. Following this incubation, the plates were washed as described above, the bound Ab was detected by the addition of o-phenylenediamine substrate (Eastman Kodak, Rochester, NY) in 20 mM citrate buffer (pH 4.0) plus 0.05% hydrogen peroxide, and the OD was read spectrophotometrically at 450 nm.
Arthus reaction
We adapted a previously described model (30) to determine whether the Arthus reactivity induced by SpA was mediated by the interaction of the Fab-binding site on SpA with reactive IgG molecules. In this model, untreated rabbits injected i.d. with SpA failed to develop inflammatory reactions at injection sites. However, animals pretreated i.v. with hIgG developed Arthus reactions at cutaneous sites that had been injected with SpA. The rabbits in our experiments were sedated with 150 mg ketamine HCl (Ketaject, Phoenix Pharmaceuticals, St. Joseph, Missouri) and 10 mg xylazine (Xylaject, Phoenix Pharmaceuticals), and their backs were shaved. The animals were rested for 48 h before injections were performed to allow any inflammation that might have occurred from the shaving to subside. After 48 h, the animals were sedated with ketamine/xylazine as described above and then injected i.v. with either 170 mg hIgG, 170 mg IgG effluent from the Mod SpA-Seph column or the Seph column, or saline (5 ml), followed by 3 to 5 ml of 0.9% saline. At 10 to 15 minutes after the i.v. injection, the dorsal skin of the rabbits was prepared with alcohol. The i.d. injections of 200 µl of 900 µg of SpA or HSA (negative control) were performed at duplicate sites. The same volume of saline was injected i.d. at duplicate sites as an additional negative control. After 8 h, the dorsal skin was examined macroscopically for erythema, edema, and hemorrhage. The animals were then sacrificed, and skin biopsies were obtained from the i.d. injection sites. The specimens were fixed in 10% buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin. The histologic procedures were performed by the Laboratory of Pathology at the University of Pennsylvania School of Veterinary Medicine (Philadelphia, PA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish the Arthus model in our laboratory, three rabbits were injected i.v. with 170 mg of hIgG and then injected i.d. with 900 µg of SpA, HSA, or saline at duplicate sites 10 to 15 min later. Three additional rabbits were pretreated i.v. with saline and then injected i.d. with the above reagents as described. All rabbits were examined grossly and then sacrificed at 8 h after the i.d. injections were administered.
No macroscopic evidence of an inflammatory response was observed at any i.d.-injected sites of saline-pretreated rabbits, as previously reported (30). By contrast, erythema and induration were both seen at the SpA-injected sites of animals pretreated i.v. with hIgG (data not shown). Such reactions were not observed at the HSA- or saline-injected sites of these hIgG-pretreated animals (data not shown).
Histologic examination of the skin biopsies from the hIgG-pretreated
rabbits revealed a prominent inflammatory cell infiltrate only at the
sites injected with SpA (Fig. 1
,
A and B). The venules were congested with
PMNs. The PMNs were also observed within the walls of the venules and
in a perivascular distribution. Several areas of hemorrhage and
diapedesis of erythrocytes were also observed. The arterioles were not
involved. Rare PMNs were observed in the lumen of the venules that were
examined at the HSA-injected sites (Fig. 1C), whereas
no inflammatory cells were observed at the saline-injected sites (data
not shown). In the saline-pretreated animals, rare PMNs were observed
in venule lumens at the SpA- (Fig. 2) and
HSA-injected sites (data not shown), while no PMNs were seen at
cutaneous sites injected with saline (data not shown).
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Depletion of VH3+ IgG molecules from hIgG
To establish that the observed inflammatory response required both
SpA and human VH3+ IgGs, the hIgG
preparation was separated into a VH3-depleted
(VH3-) fraction. To obtain this fraction (as
described in Materials and Methods), hIgG was passed
twice over a Mod SpA-Seph column, and the effluent fractions were
collected. The control IgG, containing both
VH3+ and non-VH3+ hIgGs
(VH3+/non-VH3+ hIgG,
consisted of hIgG that was passed twice over a Seph column. Following
fractionation over the Mod SpA-Seph column or the Seph column, the
binding of the effluent IgG fractions to Mod SpA and SpA was examined
by ELISAs (see Materials and Methods). As shown in
Figure 3, the effluent IgG from the Mod
SpA-Seph column had a 100-fold reduction in Mod SpA-binding IgGs as
compared with equivalent concentrations of unfractionated hIgG and the
effluent IgG from the Seph column. By contrast, IgG fractions from both
columns bound SpA to a similar degree (data not shown). This finding
demonstrates that the two types of IgG fractions contain roughly
equivalent amounts of SpA-binding IgG molecules, and that the
fractionation procedure does not lead to nonspecific IgG degradation.
Neither of the IgG fractions bound BSA-coated wells (data not shown).
Therefore, fractionation of hIgG over a Mod SpA-Seph column removed a
sufficient amount of VH3+ IgGs to use this
fraction as a VH3- population of IgGs. In
addition, passage of hIgG over a Seph column did not remove
VH3+ IgGs nor did it nonspecifically degrade
the IgG molecules.
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As shown in Figure 1
, SpA induced an Arthus reaction in rabbits
pretreated with hIgG. To determine whether this reaction occurred via a
B cell superantigenic mechanism and consequently was dependent on human
VH3+ IgGs, four groups of rabbits (two
rabbits per group) were injected i.v. with 170 mg of 1)
VH3- hIgG, 2) hIgG that had been passed over a
Seph column
(VH3+/non-VH3+ hIgG),
3) unfractionated hIgG, and 4) saline, respectively. At 10 to 15 min
after the i.v. injections, all animals were challenged i.d. at
duplicate sites with 900 µg of SpA, HSA, and saline, respectively.
After 8 h, the skin was examined grossly, and biopsies were
obtained for histologic analysis. The histologic results from
representative skin biopsies from hIgG- and saline-pretreated rabbits
are depicted in Figures 1 and 2, as described above.
Animals pretreated with
VH3+/non-VH3+ hIgG
showed erythema, edema, and hemorrhage only at the SpA-injected, i.d.
sites upon gross examination (Fig. 4,
A and B). In contrast, animals pretreated
with VH3- hIgG demonstrated no erythema or
edema at any injected sites (Fig. 4C). Thus, the
depletion of VH3+ IgGs from the hIgG
preparation abrogated the macroscopic signs of an Arthus reaction at
SpA-injected, i.d. sites.
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, A and
B). Dense infiltrates of PMNs were observed in a
perivascular distribution surrounded by areas of hemorrhage. Again, the
arterioles were not involved. The HSA-injected sites contained few PMNs
in the lumens of the venules (Fig. 5C), while no PMNs
were seen at the saline-injected sites (data not shown).
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). The rare PMNs were confined to the
lumens of the venules. The HSA-injected sites showed rare PMNs in and
around the venules, while no PMNs were seen at the saline-injected
sites (data not shown). These results indicate that SpA elicits a
prominent Arthus-like reaction in rabbits only when they are pretreated
with hIgG. Moreover, this inflammatory response is dependent upon the
presence of VH3+ IgGs in the hIgG
preparation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We chose to examine the Arthus reaction as an in vivo model of immune
complex-mediated tissue inflammation. Although this model of
inflammation was formerly thought to be mediated by complement products
liberated by the action of immune complexes (28, 29, 31), recent
studies have underscored the importance of Fc
RIII-bearing cells
(32, 33, 34). In particular, mast cells, neutrophils, macrophages, and
Langerhans cells were incriminated as critical FcRIII-bearing
effector cells (33). These studies also suggested that
complement-activation products likely augment the Arthus reaction. We
decided to investigate the Arthus-inducing potential of SpA. Unlike
other B cell SAgs, SpA has two Ig-binding sites. One site binds to the
Fc region of IgG, while the second site binds to VH region
residues on VH3+ human Igs independently of
their heavy chain isotype. It is this second site that endows SpA with
its B cell superantigenic properties.
Before the characterization of this Ig Fab-binding site on SpA, several groups investigated the similarities between conventional Ag/Ab complexes and SpA/IgG complexes (35, 36, 37, 38, 39). Indeed, one group hypothesized that SpA/IgG complexes could elicit an Arthus reaction based on the observation that Ag/Ab complexes and aggregated IgG elicit such an inflammatory reaction (30). These investigators found that i.d. injections of SpA into a rabbit did not cause an Arthus reaction. However, when the rabbits were pretreated i.v. with hIgG, an Arthus reaction occurred at SpA-injected, i.d. sites. The investigators concluded that the Arthus reaction was induced via an interaction between SpA and the Fc region of hIgG molecules. However, the authors failed to explain why an interaction with hIgG Fc, but not rabbit IgG Fc, would lead to an Arthus reaction.
One explanation for the inability of SpA to elicit an Arthus reaction
in an untreated rabbit is that the binding of SpA to residues in the
rabbit IgG Fc region blocks the binding sites for C1q and FcR. The
C1q-binding site includes residues 285 to 340 in the constant heavy
chain 2 domain of IgG Fc (40, 41, 42, 43). This region overlaps the SpA-binding
site on IgG Fc (44), although it does not bind directly to the same
residues. The FcR-binding site on IgG Fc includes residues 234 to
237 in the lower hinge region and residue 331 in the constant heavy
chain 2 domain (45, 46). These residues do not overlap the SpA-binding
site, but the binding of SpA to IgG Fc may still sterically hinder the
binding of the IgG molecule to FcR. If this explanation is correct
for rabbit IgGs, then it should also apply to hIgGs. However, SpA does
elicit an Arthus reaction when rabbits are pretreated with hIgGs. Thus,
it is unlikely that SpA blocks the binding site on IgG Fc for FcR or
C1q, at least not on hIgG molecules.
In our opinion, a more appealing explanation is that SpA only induces an Arthus reaction when it interacts with the Fab region of reactive IgGs, such as human VH3+ IgGs. SpA binds to IgG molecules from most species via their Fc and Fab regions. However, the rabbit is one of the few species in which SpA binds only to the Fc region of its IgG molecules (47, 48). Thus, it is possible that administrating SpA to a rabbit does not induce an Arthus reaction, because SpA cannot bind to the Fab region of rabbit IgGs. On the other hand, SpA elicits an Arthus reaction in rabbits pretreated with hIgGs, presumably because it can bind to the Fab region of a large proportion of hIgGs, namely VH3+ IgGs. Proof of this hypothesis would strongly suggest that a B cell SAg can trigger an in vivo immune complex-mediated inflammatory response.
Accordingly, our initial experiments were performed to confirm that SpA
elicits an Arthus reaction in rabbits pretreated with hIgG but does not
cause an Arthus reaction in untreated rabbits (30). We injected hIgG or
saline i.v. into several rabbits and then injected the rabbits i.d.
with SpA, HSA, or saline. Only animals pretreated with hIgG and
subsequently injected i.d. with SpA developed erythema and edema at
injected sites. Histologic analysis of the grossly inflamed,
SpA-injected sites revealed a prominent inflammatory response
characterized by infiltrating PMNs (Fig. 1, A–C) and
dermal hemorrhage. Animals pretreated with saline had rare luminal PMNs
only at SpA- (Fig. 2) and HSA-injected sites. Thus, these results
demonstrate that SpA that is i.d.-administered to a rabbit pretreated
with hIgG induces an inflammatory response that has the classical
histologic features of an Arthus reaction (30). In addition, our
results confirm that i.d. administration of SpA to untreated rabbits
does not lead to the development of such an inflammatory reaction.
To prove that this Arthus reaction reflected a B cell superantigenic
property of SpA, it was necessary to show that it was mediated by
VH3+ IgGs in the hIgG preparation. Therefore,
we pretreated rabbits with VH3- hIgG. The
VH3- hIgG fraction had a 100-fold reduction in
Mod SpA-binding (VH3+) IgG when compared with
unfractionated hIgG and a
VH3+/non-VH3+ hIgG
fraction which was passed over a Seph column (Fig. 3). Moreover, we
demonstrated that the VH3- hIgG fraction had
an equivalent amount of SpA-binding IgG molecules as compared with
unfractionated hIgG and the
VH3+/non-VH3+ hIgG
fraction (data not shown). The marked reduction in Mod SpA binding of
this fraction attested to the completeness of the
VH3+ IgG depletion. Following this
pretreatment, we injected the rabbits i.d. with SpA, HSA, and saline.
The cutaneous responses were compared with the reactions in animals
pretreated with
VH3+/VH3- IgG, hIgG,
or saline.
Inspection of the skin of the animals pretreated with
VH3+/non-VH3+ hIgG
revealed erythema, edema, and hemorrhage only at the SpA-injected, i.d.
sites (Fig. 4, A and B). In contrast, no
erythema or edema was observed at any injected sites from the
VH3- hIgG-pretreated animals (Fig. 4C). These results demonstrated that SpA required the
presence of human VH3+ IgGs to elicit the
macroscopic appearance of an Arthus reaction. Histologic examination of
skin biopsies from rabbits pretreated with
VH3+/non-VH3+ hIgG
demonstrated a prominent inflammatory cell infiltrate and dermal
hemorrhage only at SpA-injected sites (Fig. 5, A–C).
In striking contrast, the VH3- hIgG-pretreated
rabbits contained only minimal PMN infiltration at the SpA-injected
sites (Fig. 6). These results demonstrate that SpA is only able to
elicit a prominent inflammatory response in rabbits that are pretreated
with hIgG containing VH3+ IgGs. Even though SpA
can bind with high affinity to rabbit IgG Fc, the Arthus reaction does
not occur when human VH3+ IgGs are not present.
Therefore, an interaction between SpA, as a B cell SAg, and its
reactive hIgGs is necessary to cause tissue inflammation in
vivo.
Although the rabbit IgG does not appear to play a role by itself, it is
possible that it might affect the complexes formed between SpA and
hIgG. SpA could form complexes with IgG molecules by binding to the Fab
and Fc regions of hIgGs, in addition to the Fc region of rabbit IgG.
Such complexes would be large and possibly very stable, since SpA binds
to rabbit IgG Fc with high affinity (Ka =
109-1010 M-1) (49). The presence
of rabbit IgG might enhance the binding of these complexes to both
rabbit FcR and rabbit complement components. However, this
explanation also suggests that SpA may need to bind to both the Fab and
Fc regions of IgG molecules to cause an inflammatory response, or that
the additional binding of SpA to the rabbit IgG Fc region may not be
required but may enhance the response. Additional studies are required
to determine whether the Fc region of reactive or nonreactive IgG
molecules is needed, in addition to the Fab region of reactive IgGs, to
interact with SpA and subsequently lead to tissue inflammation in
vivo.
Nevertheless, the results described in this paper demonstrate that SpA
requires the presence of human VH3+ IgGs to
induce an Arthus reaction. It is likely that this reaction occurs via
an interaction of SpA and the Fabs of human
VH3+ IgGs. Thus, these data provide the first
evidence that the interaction of a B cell SAg, SpA, with its reactive
(VH3+) Igs leads to an inflammatory reaction in
vivo. Such an in vivo response to a B cell SAg could have profound
clinical significance. For example, SpA immunoadsorption has been used
as a novel therapy in a variety of diseases that are mediated by
pathogenic autoantibodies (50, 51). In addition, the therapy is being
investigated as a treatment for cancer, thrombotic thrombocytopenic
purpura, hemolytic uremic syndrome, myasthenia gravis, and HIV
(52, 53, 54). However, a significant number of these patients have
developed severe adverse side effects, most notably leukocytoclastic
vasculitis (52, 55). Histologically, the dermal lesions are
characterized by superficial and deep perivascular infiltrates
containing mostly neutrophils (52). In addition, diapedesis of
erythrocytes was observed. These effects could be due to an interaction
between SpA/VH3+ Igs and the formation of
immune complexes that bind to and activate FcR-bearing cells, such
as macrophages and neutrophils. Further studies will be necessary to
determine whether this type of inflammatory reaction is associated with
the in vivo response to other B cell SAgs.
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Acknowledgments |
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Footnotes |
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2 Current address: Johns Hopkins University, 755 Ross Building, 1721 E. Monument Street, Baltimore, MD 21205.
3 Address correspondence and reprint requests to Dr. Arnold Levinson, 726 Clinical Research Building, 415 Curie Boulevard, Division of Allergy and Immunology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.
4 Abbreviations used in this paper: SpA, staphylococcal protein A; VH, variable heavy chain; SAg, superantigen; HSA, human serum albumin; Mod SpA, staphylococcal protein A hyperiodinated to abrogate its IgG Fc-binding ability; Seph, CNBr-activated Sepharose 4B; hIgG, human IgG; VH3- hIgG, VH3-depleted human IgG; VH3+ using heavy chains from the VH3 gene family; VH3+/non-VH3+ hIgG, human IgG containing both VH3+ and non-VH3+ IgGs; i.d., intradermal(ly); PMN, polymorphonuclear cell.
Received for publication October 10, 1997. Accepted for publication January 29, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and the alternative F(ab')2 protein A interactions. Scand. J. Immunol. 13:343.[Medline]
-globulin. J. Immunol. 98:1178.RIII (CD16)-deficient mice. Immunity 5:181.[Medline]
-globulin. J. Immunol. 97:822. receptor. Mol. Immunol. 29:633.[Medline]
-like determinants on immunoglobulin variable regions: identification by staphylococcal protein A. Scand. J. Immunol. 15:605.[Medline]
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), or the IgG fraction from the Mod SpA-Seph column (
) were
added to Mod SpA-coated ELISA plates. Background absorbance was
determined in Mod SpA-coated wells in which no IgG proteins were
incubated, and this absorbance was subtracted from the raw data. OD
values represent the means of duplicate determinations from a
representative experiment; similar results were obtained for all
fractionated IgG preparations.
