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RI/CD89 Circulates in Human Serum Covalently Linked to IgA in a Polymeric State1
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Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands; and
Laboratory of Immunohistochemistry and Immunopathology, University of Oslo, Oslo, Norway
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI, is a type I receptor glycoprotein,
expressed on myeloid cells, with important immune effector functions.
In vitro CD89 can be released from CD89-expressing cells upon
activation. Little information is available on the existence of this
soluble molecule in vivo. Using specific and sensitive ELISA techniques
(detection limit 50 pg/ml), we were not able to detect circulating CD89
in human sera. However, using Western blotting, a 30-kDa soluble CD89
molecule was demonstrated in both serum and plasma. Moreover, using a
specific semiquantitative dot-blot system, we found CD89 in all human
sera tested (mean concentration 1900 ng/ml). Size fractionation of
human serum using gel filtration chromatography showed that the CD89
molecule was predominantly present in larger molecular mass
fractions. Direct complexes between IgA and CD89 were demonstrated by
anti-IgA affinity purification, and when analyzed under nonreducing
conditions appeared to be covalently linked. Size fractionation of
affinity-purified IgA showed the presence of soluble CD89 only in the
high molecular mass fractions of IgA, but not in monomeric IgA. High
molecular mass complexes of CD89-IgA could be distinguished from J
chain containing dimeric IgA. These data show that CD89 circulates in
complex with IgA, and suggest that CD89 might contribute to the
formation of polymeric serum IgA. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FcRI/CD89 is a type I transmembrane glycoprotein expressed at the
surface of myeloid cells and member of the Ig superfamily (2, 3). CD89 binds both IgA1 and IgA2 with similar affinity
(Ka 
106
M-1). The site of interaction between CD89 and
IgA was identified in the first extracellular domain of CD89 (4, 5) and the C2/C3 junction of IgA (6). CD89 is
constitutively expressed as a 50- to 70-kDa protein on neutrophils and
monocytes/macrophages, or as a 70- to 100-kDa glycoprotein on
eosinophils due to increased glycosylation (7). CD89 is
also expressed on Kupffer cells in the liver, where it was suggested to
provide a second line of defense (8). At the cell surface,
CD89 is associated with the FcR 
-chain through the charged arginin
residue within its transmembrane domain (9, 10). Although
it is still controversial whether this association is necessary for
surface expression (11, 12), it is critically important
for signal transduction via CD89. Cross-linking of CD89 on myeloid
cells triggers diverse processes including phagocytosis, superoxide
generation, Ab-dependent cellular cytotoxicity, and release of
inflammatory mediators (8, 13).
Release of soluble receptors is a universal mechanism of receptor
regulation and soluble forms of FcR for IgG (FcRII/CD32 and
FcRIII/CD16) and IgE (Fc
RII/CD23) have been demonstrated both in
vitro and in vivo (14). The existence of circulating
IgA-binding factors has been described (15), but their
molecular structure has remained unclear.
Recently, we showed that upon activation, a soluble form of CD89 is
released from the surface of monocytes and monocytic cell lines
(16). The molecule was identified as a 30-kDa
glycoprotein, which has still retained the capacity to bind IgA. At
present, little information is available concerning the presence of
soluble FcR/CD89 in biological fluids. In the present study, we show
by Western blot and semiquantitative dot-blot that all sera tested from
healthy volunteers contain a soluble
(s)3 CD89 molecule.
However, the molecule circulates in a complex covalently linked with
IgA in the high molecular mass fractions of serum IgA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD89 in serum was detected by ELISA as described previously (16). In short, rabbit anti-CD89 IgG (2 µg/ml) was coated to ELISA plates by overnight incubation at room temperature in coating buffer (0.1 M NaHCO3/Na2CO3, pH 9.6). Alternatively, mAbs against the extracellular domain 1 (2D11, 2H8) or extracellular domain 2 (7D7, A77) domain of CD89 (5, 17) were used as catching Ab (2 µg/ml in coating buffer). The wells were washed three times using washing buffer (PBS, 0.02% Tween 20) and samples were applied. Purified recombinant sCD89 was used as a standard curve and serum samples were tested in serial dilutions (1/2–1/100). All dilutions and subsequent Ab steps were performed in ELISA buffer (PBS, 0.02% Tween 20, 1% FCS). Following 1 h incubation at 37°C, wells were washed as above and incubated with digoxigenin-conjugated rabbit F(ab')2 anti-CD89 (1 µg/ml), followed by HRP-conjugated F(ab')2 anti-digoxigenin (1/5000; Boehringer Mannheim, Indianapolis, IN; both for 1 h at 37°C and washed in between as above). The OD415 was measured after addition of ABTS/H2O2 as substrate.
Isolation of sCD89
As a positive control, sCD89 protein was isolated from culture supernatant of PMA/ionomycin (10 ng/ml and 1 µg/ml, respectively) stimulated U937 cells using an affinity column of human IgA isolated from normal serum, as described before (16). The purity of the preparations was checked on a 10% SDS-PAGE and a single band of 30 kDa in size was detected by Coomassie brilliant blue staining.
Recombinant sCD89 (18) was produced in Chinese hamster ovary cells and purified similar to the natural sCD89 molecule (16).
Western blot analysis
Purified or recombinant sCD89, as well as serum and plasma samples obtained from healthy individuals, were separated on 10% SDS polyacrylamide gels under reducing conditions, and blotted onto polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). In all cases, 30 µl sample was mixed with 15 µl 3x sample buffer (Bio-Rad Laboratories, Hercules, CA) and boiled for 5 min. For comparison, several formulations of prestained broad range molecular mass markers (Bio-Rad) were run on the same gels. Using standard Western blotting protocols, different forms of CD89 were detected with rabbit IgG anti-CD89 (10 µg/ml; Ref. 16). After incubation and washing followed by incubation with HRP-conjugated swine anti-rabbit-IgG (1/50,000; DAKO, Glostrup, Denmark), signals were visualized using Super Signal Chemiluminescence substrate, according to manufacturer’s instructions (Pierce, Rockford, IL).
Alternatively, samples were analyzed under nonreducing conditions on either 10 or 6% SDS-PAGE gels, or precast 4–15% gradient gels (Bio-Rad). They were analyzed by Western blotting using the monoclonal anti-CD89 Ab 7D7 (IgG1) at 10 µg/ml, followed by HRP-conjugated goat anti-mouse-Ig (DAKO) and chemiluminesence as described above.
IgA was detected in Western blot using the monoclonal anti-IgA Ab 4E8 (IgG1) at 2 µg/ml (19). J chain was detected in Western blot using a J chain-specific rabbit polyclonal antiserum (20, 21).
Detection of sCD89 by dot-blot technique
Sera were diluted 1/100, 1/1,000, and 1/10,000 in PBS and blotted onto a PVDF membrane using a special dot-blot device (Bio-Rad), according to manufacturer’s instructions. Alternatively, fractions obtained from gel filtration columns were blotted. Membranes were developed for CD89 reactivity with the polyclonal anti-CD89 antiserum as described for Western blotting. As a standard, we included purified sCD89 molecule derived from U937 cells on every blot, in a concentration range starting from 10 ng/ml. For specificity control, the anti-CD89 antiserum was preincubated with 25 µg/ml recombinant sCD89 produced in Chinese hamster ovary cells, which prevented specific binding. Densitometry was performed using the EagleEye software package (version 3.2; Stratagene, San Diego, CA).
Fractionation of serum
Randomly selected sera from healthy controls (n = 6) were separated by size on a 1.5 x 90 cm Sephacryl S300 column (Amersham Pharmacia, Roosendaal, The Netherlands). Briefly, 0.5-ml aliquots of sera were diluted with an equal volume of PBS layered on the column, and fractions of 1.25 ml were collected. The fractions were analyzed for protein content using BCA protein assay (Pierce), and CD89 using ELISA- and CD89-specific blotting.
IgA affinity purification and fractionation
A total of 50 µl of serum was diluted with 950 µl of PBS-EDTA, mixed with anti-IgA immunoabsorbent (mAb 4E8-Sepharose, produced in our laboratory), rotated at 4°C overnight, and subsequently poured in a small column. The fall-through was collected, followed by extensive washing with PBS-EDTA. Bound IgA was eluted with 0.1 M glycine-HCl, 0.3 M NaCl (pH 2.8), and neutralized by 15 µl Tris buffer per milliliter.
Purified IgA, or total serum as comparison, were separated by size on a 26/60 HR200 Superdex column (Amersham Pharmacia) and fractions of 2 ml were collected. The fractions were analyzed for protein content using BCA protein assay, total IgA using ELISA (sensitivity 2 ng/ml; Ref. 19), and CD89 using ELISA and CD89-specific blotting.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the presence of sCD89 molecules in circulation,
Western blotting was performed on samples separated by SDS-PAGE under
reducing conditions, as described before. In both serum (Fig. 1
) and plasma, a 30-kDa CD89 molecule
could be visualized. This band has a molecular mass which corresponds
with the sCD89 molecule released by U937 cells after activation with
PMA/ionomycin (Fig. 1, lane 1; Ref. 16).
Preincubation of the detecting Ab with rCD89 (25 µg/ml) completely
abrogated the signal, proving specificity of this Western blot
detection (Fig. 1, lanes 6–9).
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To quantify the amount of circulating CD89 in human serum, we used
different CD89-specific ELISAs based on polyclonal reagents of rabbit
origin (16), or using mAbs directed against the
extracellular domains 1 or 2 of CD89. We tested serial dilutions
(1/2–1/100) of serum or plasma from 48 healthy volunteers. None of the
samples showed reactivity in any of these CD89-specific ELISAs (Figs. 2 and 3B). In contrast, sCD89
present in supernatant of PMA/ionomycin-activated U937 cells could be
detected with both polyclonal and mAbs as catching Ab (Fig. 2).
Moreover, the same ELISAs were able to detect recombinant sCD89 added
to human serum (data not shown).
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Because Western blotting revealed a single 30-kDa band which was
completely inhibited by preincubation with recombinant sCD89, we have
set up a dot blot system specific for CD89, using the same reagents.
Using purified sCD89 from PMA/ionomycin activated-U937 as a standard,
dose-dependent increase in dot blot reactivity was observed (Fig. 3
A). A similar dose-response curve was obtained when
applying different dilutions of normal human serum (NHS). In both
cases, all reactivity could be inhibited by preincubation of the
antiserum with recombinant sCD89 (25 µg/ml; Fig. 3A).
Using a dose response of purified sCD89 on every dot blot, the amount
of sCD89 in human serum was quantified using densitometry. In the same
panel of serum samples of healthy controls (n = 48)
which were negative in ELISA (<50 pg/ml), we found the presence of
CD89 in all cases tested. The estimated mean value of CD89 in these
sera was 1900 ng/ml (Fig. 3B).
CD89 circulates in a high molecular mass complex
To study the molecular nature of CD89 in human serum, we
fractionated sera using a Sephacryl S300 column and tested the
fractions for CD89. Using the dot-blot technique, we could only detect
CD89 in the high molecular mass range above 150 kDa, fraction volumes
from 105 to 155 ml, but not in fraction volumes 205–210 ml,
where 30-kDa proteins are expected (Fig. 4A). None of the fractions
showed reactivity in the CD89 ELISA (data not shown). A similar profile
was found with six independent sera.
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B). The same 30-kDa band was observed when the Western blot
was performed with the monoclonal anti-CD89 Ab 7D7 instead of the
polyclonal antiserum (Fig. 4C). Interestingly, samples run
under nonreducing SDS-PAGE conditions revealed CD89 reactivity around
180 kDa, when Western blots were stained with this 7D7 mAb. Affinity purification of IgA-CD89 complexes
Since we previously showed that sCD89 was still able to bind IgA
(16), specific interaction with IgA was the prime
candidate to explain the high molecular mass appearance of CD89.
Therefore, IgA was purified from serum samples using anti-IgA
affinity purification. IgA was purified from human serum with a
recovery of 95%, and was pure as judged by Coomassie blue staining
(data not shown). Size fractionation of this purified IgA showed a
complete overlap of the IgA profile and the protein content (Fig. 5A, right panel).
The size distribution of purified IgA was comparable to total serum
(Fig. 5A, left panel).
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B). These data
confirm the presence of IgA-CD89 complexes. CD89 is exclusively present in circulating dimeric and polymeric IgA
In circulation, IgA is present in different molecular sizes being
either monomeric, dimeric, or polymeric in nature. To determine whether
sCD89 preferentially associates with certain IgA types, IgA was
isolated from serum using the above described affinity purification,
followed by size fractionation. Measurement of IgA in these fractions
using ELISA showed the typical distribution of IgA, with the majority
being monomeric (Fig. 6A).
When the same fractions were analyzed by CD89-specific dot blot,
reactivity was most pronounced for dimeric and polymeric IgA, and
almost absent from the monomeric fraction.
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B). The dimeric IgA fraction
consisted of two major bands, one representing dimeric IgA and one
migrating slightly faster as monomeric IgA. Finally, IgA circulating in
a higher molecular mass form (polymeric) showed a similar pattern as
dimeric IgA fractions.
Next, the same samples were analyzed for CD89. The reactivity for CD89
was found at the height of the lower molecular mass IgA molecule, but
exclusively in these fractions which appear as higher molecular mass in
gel filtration (Fig. 6B).
IgA-CD89 and IgA-J chain complexes can be distinguished in dimeric serum IgA
To further investigate the molecular nature of the two differently
sized IgA molecules in the dimeric IgA fraction, we performed Western
blotting with a specific anti-J chain antiserum (20, 21). Anti-J chain specifically detected the upper 350-kDa
dimeric IgA band in Western blot, but not the CD89-IgA complex (Fig. 7). IgA, truly monomeric both in gel
filtration and Western, was completely negative for J chain or for
CD89, even when larger amounts of IgA were loaded (Fig. 7). Therefore,
high molecular mass serum IgA might be either formed by covalently
linked, J chain containing, dimeric IgA, or by multimerization of
covalent IgA-CD89 complexes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Covalent linkage between IgA and the soluble form of a cell surface receptor is not unique for the IgA-CD89 complex, as described in the present paper. Dimeric IgA containing J chain uses binding to the polymeric IgR (pIgR) for transcytosis from the basal to the apical site of epithelial cells in the mucosa (22). During a late phase of the transcytosis process, IgA becomes covalently linked to the pIgR (23, 24). At the apical membrane, this receptor is proteolytically cleaved, and IgA is released as secretory IgA (sIgA), retaining the so-called secretory component (25). Mice lacking the J chain are deficient in the formation of sIgA (26), and have a phenotype comparable to pIgR-deficient mice (27). Interestingly, J chain knockout mice do show the formation of higher molecular mass IgA in serum, confirming a multimerization of IgA in the absence of J chain. This might suggest the involvement of other molecules, but since a murine CD89 homolog has not yet been identified, in these cases the role of CD89 cannot be addressed.
Our data show that J chain-containing dimeric IgA is completely negative for CD89. Therefore, it can be hypothesized that J chain and CD89 are competing for the same cysteine. In our Western blot experiments, we could clearly distinguish CD89-IgA complexes and J chain-containing dimeric IgA. However, this does not mean that dimeric IgA cannot interact with cell surface CD89, as demonstrated before. At the moment, we favor the hypothesis that the site of covalent linkage can occur at a different location from the initial binding site, in line with dIgA-pIgR interaction and the generation of sIgA (28).
Our study suggested a discrepancy between detection of circulating CD89 in ELISA or dot-blot. However, this difference can be explained by the observed covalent interaction between CD89 and IgA. CD89 consists of two Ig domains, both formed by a single intradomain disulfide bond (2). Therefore, the covalent linkage to IgA will have dramatic structural consequences. This is in contrast to the formation of sIgA, where the fifth Ig domain, which becomes covalently linked to IgA, contains three intradomain disulfide bonds, allowing one bond to be used for interaction with IgA (28). This most likely explains why CD89-IgA complexes cannot be detected by ELISA, even when the amount of CD89 estimated by dot-blot is many orders higher than the detection limit of the ELISA.
An important open question is the mechanism of complex formation. In
analogy with the pIgR system, it is most likely that reshuffling of
cysteine bonds takes place in an intracellular compartment. Previously,
we have shown that the common -chain is necessary for the generation
of the 30-kDa CD89 molecule and that IgA is a potent inducer of
shedding in monocytic cells, but not neutrophils (16).
Others have suggested that CD89 can exist both in the absence or
presence of the -chain (12). In the absence of
-chain, IgA which binds to CD89 is internalized and localizes mainly
in early endosomes. However, in the presence of -chain, CD89 and
bound ligands are internalized and transported into an endolysosomal
compartment (12). Especially in cells like monocytes, such
a compartment is known to have special features (29).
Therefore, this could be a potential mechanism by which monomeric IgA
becomes covalently linked with CD89. Although induction of signal
transduction via CD89 is more efficent with high molecular mass IgA
complexes (30), Biacore experiments have clearly
demonstrated that monomeric IgA can also efficiently interact with CD89
(31).
Recently, complexes of CD89 and IgA have been described in serum of
patients with IgA nephropathy (32). In this study,
sCD89 could be detected by ELISA, although only after polyethylene
glycol precipitation. In contrast to our previous data
(16), the sCD89 molecule described was spontaneously
released from the surface of monocytes in vitro. This shedding was
independent of signaling via the -chain, and the molecule appeared
to have a different molecular mass (32). Moreover, sCD89
was only detected in patients with IgA nephropathy, but not in healthy
controls. In preliminary experiments, we have observed that covalent
IgA-CD89 complexes are equally present in both groups (P. van der Boog,
manuscript in preparation). These data strongly suggest that
CD89 can circulate in different molecular forms, either low
concentrations of free or IgA-bound CD89 (ELISA detectable) and high
amounts of covalent CD89-IgA complexes.
The pathophysiological role of IgA-CD89 complexes is presently unknown.
Soluble forms of other FcR, specific for IgG or IgE, have been
extensively described in the past. Circulating molecules with
IgE-binding capacity (IgE-binding factors) were identified as the
soluble form of FcRII (sCD23; Refs. 33 and
34), and play an important role in the regulation of IgE
production. In NHS, low levels of FcRII (sCD32; 10 ng/ml) and
higher levels of sFcRIII (sCD16; 2.5 µg/ml) have been
demonstrated (14). These levels of sCD16 are in the same
range as what we observed for circulating CD89. Release of soluble
receptors will terminate the ongoing signaling process and might
represent a mechanism to prevent prolonged activation. Moreover,
release of soluble receptors that still retain their ligand binding
capacity will also help to terminate the signaling process. The latter
is most likely not the case for the circulating CD89 described in the
present paper, since it is already associated with IgA, and the
structure of the protein is most likely disturbed. Alternatively,
facilitation of the formation of high molecular mass IgA by sCD89 might
result in more efficient binding and cross-linking of the cell surface
CD89 receptor. Therefore, it will be important to investigate and
compare the effector functions of IgA and IgA complexed with CD89,
including binding to other receptors, clearance in vivo, or resistance
to proteolysis.
In conclusion, we have demonstrated that a part of serum IgA consists of IgA-CD89 complexes. CD89 and monomeric IgA form a covalently linked complex, which seems to facilitate multimerization to polymeric serum IgA and circulates in high molecular mass fractions. We have estimated that up to 5–10% of serum IgA might be complexed with CD89, but more detailed studies are needed to confirm this. By Western blot, the CD89-IgA complex can be distinguished from J chain-containing dimeric IgA. It will be important to determine the effector functions of these CD89-IgA complexes, which might add another complexity to the role of IgA in both mucosal and systemic immunity.
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Footnotes |
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2 Address correspondence and reprint requests to: Dr. Cees van Kooten, Department of Nephrology, Leiden University Medical Center, C3P, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. E-mail address: kooten{at}lumc.nl
3 Abbreviations used in this paper: s, soluble; PVDF, polyvinylidene difluoride; pIgR, polymeric IgR; sIgA, secretory IgA; NHS, normal human serum.
Received for publication May 7, 2001. Accepted for publication December 4, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor in humans. J. Exp. Med. 171:597. receptor essential for interaction with IgA. J. Immunol. 162:2146.R, CD89) and bovine Fc2R to their membrane-distal EC1 domains. J. Exp. Med. 189:1715.2 and C3 in human IgA1. J. Exp. Med. 183:1579.RI-positive liver kupffer cells: reappraisal of the function of immunoglobulin A in immunity. Nat. Med. 6:680.[Medline]
R) with FcRI2 subunits in U937 cells: aggregation induces the tyrosine phosphorylation of 2. J. Immunol. 153:3228.[Abstract]
chain: molecular basis for CD89/FcR chain association. J. Biol. Chem. 270:29781.RI, CD89) function in transgenic mice requires both FcR chain and CR3 (CD11b/CD18). Blood 93:4387. association and protects against degradation of bound ligand. J. Biol. Chem. 274:7216.R). Crit. Rev. Immunol. 16:423.[Medline]
receptors: interaction with ligands and biological consequences. Int. Rev. Immunol. 16:87.[Medline]
chain-dependent shedding of a soluble form of CD89. J. Immunol. 163:5806. receptor (CD89). J. Am. Soc. Nephrol. 10:770.RI with IgA and its implications for ligand binding by immunoreceptors of the leukocyte receptor cluster. J. Immunol. 166:1781. receptor (CD89) mediates the development of immunoglobulin A (IgA) nephropathy (Berger’s disease): evidence for pathogenic soluble receptor-IgA complexes in patients and CD89 transgenic mice. J. Exp. Med. 191:1999.This article has been cited by other articles:
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C. Pilette, K. T. Nouri-Aria, M. R. Jacobson, L. K. Wilcock, B. Detry, S. M. Walker, J. N. Francis, and S. R. Durham Grass Pollen Immunotherapy Induces an Allergen-Specific IgA2 Antibody Response Associated with Mucosal TGF-beta Expression J. Immunol., April 1, 2007; 178(7): 4658 - 4666. [Abstract] [Full Text] [PDF]
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 B. D. Oortwijn, A. Roos, L. Royle, D. J. van Gijlswijk-Janssen, M. C. Faber-Krol, J.-W. Eijgenraam, R. A. Dwek, M. R. Daha, P. M. Rudd, and C. van Kooten Differential Glycosylation of Polymeric and Monomeric IgA: A Possible Role in Glomerular Inflammation in IgA Nephropathy J. Am. Soc. Nephrol., December 1, 2006; 17(12): 3529 - 3539. [Abstract] [Full Text] [PDF]
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 B. D. Wines, H. M. Trist, P. A. Ramsland, and P. M. Hogarth A Common Site of the Fc Receptor {gamma} Subunit Interacts with the Unrelated Immunoreceptors Fc{alpha}RI and Fc{epsilon}RI J. Biol. Chem., June 23, 2006; 281(25): 17108 - 17113. [Abstract] [Full Text] [PDF]
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A. Roos, M. P. Rastaldi, N. Calvaresi, B. D. Oortwijn, N. Schlagwein, D. J. van Gijlswijk-Janssen, G. L. Stahl, M. Matsushita, T. Fujita, C. van Kooten, et al. Glomerular Activation of the Lectin Pathway of Complement in IgA Nephropathy Is Associated with More Severe Renal Disease J. Am. Soc. Nephrol., June 1, 2006; 17(6): 1724 - 1734. [Abstract] [Full Text] [PDF]
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 P. J. M. van der Boog, C. van Kooten, A. van Seggelen, M. Mallat, N. Klar-Mohamad, J. W. de Fijter, and M. R. Daha An increased polymeric IgA level is not a prognostic marker for progressive IgA nephropathy Nephrol. Dial. Transplant., October 1, 2004; 19(10): 2487 - 2493. [Abstract] [Full Text] [PDF]
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) as well as CD89 reactivity using
dot-blot (
). The molecular mass of the qualibrated column are shown
on top of the figure. One representative fractionation out of six is
shown. B, Starting material (NHS; 1/50 dilution), high
(110, 130, and 150 ml) and low (215 and 200 ml) volume
fractions (30 µl) were analyzed by Western blot as described in Fig. 1
) and IgA (•) as described in
Materials and Methods. The molecular mass of the
qualibrated column are shown on top of the figure. B,
Two dilutions (1/80 and 1/160) of starting material (NHS), eluate, or
fall-through of an IgA affinity column were analyzed under nonreducing
conditions as described above. For comparison, the amount of eluate was
corrected for the change in volume. One of six purifications is
shown.
