Báo cáo Y học: Human immunoglobulin A (IgA)-specific ligands from combinatorial engineering of protein A ppt

Human immunoglobulin A (IgA)-specific ligands from combinatorial
engineering of protein A
Jenny Ro¨ nnmark
1
, Hans Gro¨ nlund
2
, Mathias Uhle
´
n
1
and Per-A
˚
ke Nygren
1
1
Department of Biotechnology, Royal Institute of Technology (SCFAB), Stockholm, Sweden;
2
Department of Medicine,
Unit of Clinical Immunology and Allergy, Karolinska Institute, Sweden
Affinity reagents capable of selective recognition of the dif-
ferent human immunoglobulin isotypes are important
detection and purification tools in biotechnology. Here we
describe the development and characterization of affinity
proteins (affibodies) showing selective binding to human
IgA. From protein libraries constructed by combinatorial
mutagenesis of a 58-amino-acid, three-helix bundle domain
derived from the IgG-binding staphylococcal protein A,
variants showing IgA binding were selected by using phage
display technology and IgA monoclonal antibodies (mye-
loma) as target molecules. Characterization of selected
clones by biosensor technology showed that five out of eight

investigated affibody variants were capable of IgA binding,
with dissociation constants (K
d
) in the range between 0.5 and
3 l
M
. One variant (Z
IgA1
) showing the strongest binding
affinity was further analyzed, and showed that human IgA
subclasses (IgA
1
and IgA
2
) as well as secretory IgA were
recognized with similar efficiencies. No detectable cross-
reactivity towards human IgG, IgM, IgD or IgE was
observed. The potential use of the Z
IgA1
affibody as a ligand
in affinity chromatography applications was first demon-
strated by selective recovery of IgA protein from a spiked
Escherichia coli total cell lysate, using an affinity column
containing a divalent head-to-tail Z
IgA1
affibody dimer
construct as a ligand. In addition, efficient affinity recovery
of IgA from unconditioned human plasma was also dem-
onstrated.
Keywords: IgA; affibody; combinatorial protein engineering;

affinity ligand; phage display.
Efficient and selective methods for immunoglobulin (Ig)
detection and purification are of major importance for a
vast number of applications within areas such as immuno-
logy, diagnostics and biotechnology. For these purposes, an
often recruited class of reagents is derived from so-called
receptin proteins, produced as surface-anchored or soluble
proteins by some bacteria [1]. Many of these proteins show
binding to one or more mammalian serum or cell surface
proteins, including for example Ig, serum albumin and
fibrinogen [1]. One of the most well-known Ig-binding
receptins is staphylococcal protein A, widely used in many
formats for its capability to bind a wide spectrum of Igs via
Fc or V
H
region recognition [2–5]. Other examples of
frequently used Ig-binding receptins include streptococcal
protein G [6,7] and Peptostreptococcus magnus protein L
[8,9]. The binding specificities displayed by different
Ig-binding receptins differ significantly in terms of Ig
isotype, subclass, species origin and subfragment type (Fc,
Fab, scFv, etc.), which makes the choice of reagent for a
particular application important.
IgA is the most abundant Ig isotype in humans where it is
present in two subclasses, IgA
1
and IgA
2
, differing mainly in
their hinge region sequences [10]. IgA is predominantly

localized to mucosal surfaces but is also present at high
levels in plasma [11] and believed to play an important role
in defence mechanisms against infections [12]. IgA is also
implicated in several diseases including IgA nephropathy
[13], coeliac disease [14], Henoch-Scho
¨
nlein purpura [15]
and neurological diseases [16], where IgA levels are mon-
itored as a disease marker or investigated for a more direct
involvement in disease progression. Interestingly, recent
data suggest that IgA should be an interesting isotype to
validate for development of antibodies for immunotherapy
applications, including cancer therapy, due to its effective
recruitment of neutrophils [17].
The identification of receptins or the development of
other classes of ligands capable of selective IgA binding will
therefore be important for biotechnology tools, facilitating
IgA recovery and detection. Interestingly, receptin proteins
shown to be capable of IgA binding have been described,
including the Sir22 and Arp4 proteins isolated from
Streptococcus pyogenes [18,19]. These proteins were recently
shown to bind both IgA
1
and IgA
2
and mapped to
recognize the CH
2
–CH
3

interface on the IgA Fc fragment.
The biotechnological use of this class of proteins has been
recognized and initially investigated for immunodetection
applications using the IgA binding protein B from group B
streptococci [20].
In this work, we describe an alternative approach to
obtain novel IgA binding ligands, using combinatorial
protein engineering to change the binding specificity of an
already existing receptin protein. Employing a 58-amino-
acid domain derived from one of the immunoglobulin
binding (IgG) domains of staphylococcal protein A as a
scaffold, we have previously described the construction of
Correspondence to P A
˚
. Nygren, Department Biotechnology, Royal
Institute of Technology, SCFAB, SE-106 91 Stockholm, Sweden.
Fax: + 46 855378481, Tel.: + 46 855378328,
E-mail:
Abbreviations: c.f.u., colony forming units; K
d
, dissociation constant;
ABD, albumin binding domain; HSA, human serum albumin; NHP,
normal human plasma; CIC, circulating immune complexes.
(Received 31 January 2002, revised 5 April 2002,
accepted 10 April 2002)
Eur. J. Biochem. 269, 2647–2655 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02926.x
combinatorial protein libraries from which novel affinity
proteins (denoted Z-affibodies) have been selected for
binding to desired target proteins using phage display
technology [21,22]. In common with their protein A

ancestor, several of the described affibodies have been
shown to be useful as selective and robust ligands in affinity
chromatography applications [23–25]. Here, we describe the
use of the affibody technology platform for the identifica-
tion of protein A-derived variants showing IgA- rather than
IgG-binding, thus expanding the available repertoire of
tools for detection and recovery of native or recombinant
IgA from different sources. The selection procedure,
biosensor binding affinity and specificity data of candidate
ligands as well as the affinity chromatographic recovery of
IgA from a bacterial lysate and unconditioned human
plasma is described.
MATERIALS AND METHODS
Strains and vectors
E. coli strain RR1DM15 (supE) [26] was used as host for the
phage production and cloning work. For protein expres-
sion, strains RRIDM15 or the nonsuppressor E. coli strain
RV308 [27] were used, as indicated. The phagemid vector
pKN1 has been described previously [21,22]. Phage stocks
were prepared using M13K07 helper phage (New England
Biolabs).
Affibody phage display libraries
The construction of the Z-affibody phage libraries Zlib-1
and Zlib-2 has been described previously [21,22]. The two
libraries differ in the use of degeneracy for variegated
codons, where NN(G/T) and (C/A/G)NN codons were
used for Zlib-1 and Zlib-2, respectively. Both libraries have
sizes of  4.5 · 10
7
members.

Phage stocks
The phage stocks were prepared as follows. Cells hosting the
respective phagemid library were cultivated in shake flasks
containing 50 mL tryptic soy broth supplemented with
yeast extract (TSB+YE) and ampicillin (100 lgÆmL
)1
) at
37 °CtoD
600
¼ 0.5. An aliquot (10 mL) was incubated
with 10
11
colony forming units (c.f.u.) of M13K07 helper
phage for 30 min at 37 °C followed by centrifugation at
2500 g for 10 min The cells were resuspended and trans-
ferred to 100 mL TSB+YE with ampicillin (100 lgÆmL
)1
),
kanamycin (25 lgÆmL
)1
) and isopropyl thio-b-
D
-galacto-
side (100 l
M
) and cultivated overnight ( 20 h) at 30 °C.
The supernatant of the overnight culture was subjected to
poly(ethylene glycol) precipitation: 25 mL poly(ethylene
glycol)/NaCl [20% poly(ethylene glycol) 6000, 2.5
M

NaCl]
was added and the mixture was incubated on ice for
 60 min at 4 °C. After centrifugation at 10 400 g for
30 min, the pellet was resuspended in 15 mL distilled water
and another 2.75 mL poly(ethylene glycol)/NaCl was added
for a second precipitation on ice for 30 min at 4 °C followed
by centrifugation at 16 300 g for 30 min. Finally, the pellet
was resuspended in NaCl/P
i
(50 m
M
phosphate, 100 m
M
NaCl, pH 7.2) and filtered (0.45 lm). The phage library
titers were calculated to 1 · 10
11
and 1 · 10
12
c.f.u. per mL
for Zlib-1 and Zlib-2, respectively, from a serial dilution of
phages allowed to infect E. coli andplatedonagarplates
containing ampicillin (100 lgÆmL
)1
).
Selections
Two different human myeloma IgA
1
antibody prepara-
tions, denoted IgA1116 and IgA2167 (kind gifts from
Pharmacia Diagnostics AB, Sweden), were used as panning

targets during selections. The IgA antibodies were sepa-
rately biotinylated in vitro using a
D
-biotinoyl-e-amidocap-
roic acid N-hydroxysuccinimide ester (Boehringer
Mannheim GmbH, Germany), according to the supplier’s
recommendations. Biotinylated antibodies were bound to
streptavidin-coated paramagnetic DynabeadsÒ (SA-beads)
(Dynal AS, Norway) at conditions resulting in  30 lgIgA
per mg beads. In each selection cycle, approximately
10
10
)10
11
phages were mixed with 5 mg target-containing
beads in a total volume of 100 lL, resulting in a final IgA
concentration during selection of approximately 10 l
M
.
Prior to selection, IgA-containing beads were incubated
with 0.1% gelatine for 30–60 min Prior incubation with
IgA-coated beads phage stocks were incubated with clean
SA-beads (cycles two to five). The two IgA
1
antibodies
were used alternatively as targets during selections
(IgA2167: cycles 1, 2 and 4; IgA1116: cycles 3 and 5).
Phage stocks were incubated with target-containing beads
for 2–4 h, after which the beads were washed with portions
of 500 lLofNaCl/P

i
buffer according to the following.
Cycle 1: two washes (10 min); cycles 2–4: six washes
(25 min); cycle 5: 10 washes (27 min). Bound phages were
released with 500 lLof0.1
M
glycin/HCl pH 2.2 for
10 min and the supernatant was neutralized in 50 lL1
M
Tris/HCl pH 8.5. All steps were performed at room
temperature (20–22 °C) and all tubes were preblocked
with NaCl/P
i
supplemented with 0.1% gelatine. The two
eluates originating from separate use of the Zlib-1 and Zlib-
2 libraries in the first cycle were mixed and treated as one
library in the following cycles. The selection process was
monitored by titrating the phage stocks before selection
and after elution as described above.
DNA sequencing
Randomly picked colonies after infection with the cycle five
eluate were subjected to PCR amplification of inserts for
solid phase DNA sequencing using an ALFexpress
TM
sequencing instrument (Amersham Biosciences, Sweden),
as described previously [21]. Other constructs were
sequenced by cycle sequencing DNA methodology on a
MegaBACE
TM
1000 instrument (Amersham Biosciences)

using BigDye Terminators (PerkinElmer Applied Biosys-
tems, USA).
Production and purification of proteins from phagemid
constructs
Sequenced clones were expressed as periplasmically secreted
affibody-ABD fusion proteins in the nonsuppressor E. coli
strain, RV308. From an overnight culture in the same
medium, 1 mL was inoculated to 100 mL TSB+YE
supplemented with ampicillin (100 lgÆmL
)1
) and cultivated
at 37 °CtoaD
600
of  1. This culture was induced by
2648 J. Ro
¨
nnmark et al. (Eur. J. Biochem. 269) Ó FEBS 2002
adding isopropyl thio-b-
D
-galactoside to a final concentra-
tion of 1 m
M
and cultivated at room temperature overnight.
The periplasmic fraction was collected by an osmotic shock
procedure and the clones were purified by affinity chroma-
tography on human serum albumin Sepharose column as
follows: the column was pulsed with 0.5
M
HAc, pH 2.8 and
TST (25 m

M
Tris/HCl, 200 m
M
NaCl, 1.25 m
M
EDTA,
0.05% Tween 20, pH 8.0), respectively, and equilibrated
with TST before loading the sample. After washing with
TST, pH was lowered by adding 5 m
M
NH
4
Ac (pH 5.5)
and the protein was eluted with 0.5
M
HAc, pH 2.8.
Relevant fractions were lyophilized, separated on a homo-
genous 20% SDS/PAGE PhastGel
TM
(Amersham Bio-
sciences) and stained with Coomassie Brilliant Blue.
Biosensor analyses
ABiacore
TM
2000 (Biacore, Sweden) was used for the
biospecific molecular interaction studies between selected
affibodies and IgA2167. As running buffer, HBS (10 m
M
Hepes, 150 m
M

NaCl, 3.4 m
M
EDTA, 0.005% Surfactant
20, pH 7.4) was used at a flow rate of 5 lLÆmin
)1
in all
experiments. Between injections, the surfaces were regene-
rated with 10 m
M
HCl and/or 0.05% SDS. Proteins were
immobilized on CM5 chip surfaces (Biacore) using amine
coupling chemistry according to the manufacturer’s
recommendations. At one chip,  3400 RU (response
units) of HSA and 5300 RU of IgA2167 were immobilized
in different flow cells. Initial analyses of selected variants
were performed using the HSA surface by injection of
25 lL (25–70 l
M
) protein dissolved in HBS, directly
followed by injection of 25 lL IgA2167 (2 l
M
) in HBS.
The IgA2167 surface was used for binding affinity studies
through steady state response value analyses for different
concentrations of injected analyte employing
BIAEVALUA-
TION
3.0 software (Biacore). The program fits the data to
the formula K
a

¼ R
eq
/(R
max
) R
eq
) where R
eq
is the
equilibrium response at a given concentration of analyte.
K
a
( ¼ 1/K
d
) is the affinity constant (
M
)1
) and R
max
the
response at saturation. Three affibody variants were
investigated with random injection (duplicate samples) of
25 lL protein with concentrations ranging from 40 n
M
to
140 l
M
. Regeneration was performed with 5 lL0.05%
SDS.
One chip was prepared with Z

IgA1
-ABD protein
(720 RU), Z
wt
-ABD protein (640 RU) and ABD protein
(reference, 380 RU), immobilized in different flow cells for
initial IgG/IgA binding specificity studies and the same chip
was also used for subsequent human isotype and subclass
specificity analyses. 35 lLsamples(0.2l
M
) of human
polyclonal IgA (Sigma–Aldrich Chemie, Germany, cat. no
I-1010), human polyclonal IgA
1
(Calbiochem, USA, cat.
no. 400105), human myeloma IgA
2
(Calbiochem, cat.
no. 400110), human myeloma IgM (Pharmacia Diagnos-
tics), human polyclonal IgG (Pharmacia, Sweden), human
myeloma IgD (Chemicon, USA, art. no AG740), secretory
IgA (Nordic Immunological Laboratories, Netherlands,
cat. no P020) and a monoclonal recombinant IgE
mouse::human chimera consisting of murine light chain
and VH domains and human epsilon 1–4 heavy chain
domains [28] were separately injected and the responses
recorded. The reference surface (ABD) was used to produce
subtractive sensorgrams.
Construction and production of dimeric (head-to-tail)
affibody constructs

The gene fragment encoding the Z
IgA1
affibody was
amplified from a pKN1-Z
IgA1
phagemid template by PCR
using primers NOKA-6 and NOKA-7 [29]. The resulting
PCR product was cleaved with SalIandXhoI and ligated
into the XhoI site (in front of the ABD tag) of the phagemid
pKN1-Z
IgA1
. The resulting construct denoted pKN1-dZ
IgA1
thus encodes a dimeric (head-to-tail) (Z
IgA1
)
2
-ABD affibody
construct. This construct was subsequently further modified
through the insertion of a linker sequence formed by the
oligonucleotides ZlibHisStop-1: 5¢-TCGACCCATCAT
CATCATCATCATTAATAAGTCGAC-3¢ and ZlibHis-
Stop-2: 5¢-TCGACGTCGACTTATTAATGATGATGA
TGATGATGATGGG-3¢ encoding a hexahistidyl sequence
followed by a stop codon into the XhoI site (in front of the
ABD tag), resulting in phagemid pKN1-dZ
IgA1
-His
6
enco-

ding a dimeric (head-to-tail) (Z
IgA1
)
2
-His
6
affibody con-
struct. The (Z
IgA1
)
2
-ABD affibody fusion protein encoded
by phagemid pKN1-dZ
IgA1
was produced in strain RV308
and purified as described above. The (Z
IgA1
)
2
-His
6
affibody
fusion protein was produced in strain RR1DM15 under the
same conditions as above and purified from the periplasm
by immobilized metal ion affinity chromatography (IMAC)
using 2 mL TALON
TM
(Co
2+
) media (Clontech Laborat-

ories Inc., CA, USA). After a standard osmotic shock
procedure, the periplasmic fraction was dissolved in load-
ing/running buffer (50 m
M
NaH
2
PO
4
,10m
M
Tris/HCl,
100 m
M
NaCl, pH 8.0) and loaded on the TALON
TM
column, pre-equilibrated with 20 mL running buffer.
The column was washed with 30 mL running buffer
and the protein was released with 10 · 1mL elution
buffer (50 m
M
NaH
2
PO
4
,100 m
M
HAc, 100 m
M
NaCl,
pH 5.0). Fractions 3–10 were desalted on a PD

TM
-10
column (Amersham Biosciences) with a simultanous
buffer change to 5 m
M
NH
4
Ac pH 5.5, lyophilized and
than analysed on a homogenous 20% SDS/PAGE
PhastGel
TM
(Amersham Biosciences) stained with Coomas-
sie Brilliant Blue.
Ligand immobilization and affinity chromatography
The (Z
IgA1
)
2
-ABD or (Z
IgA1
)
2
-His
6
affibody fusion proteins
(2.5 mg and 2 mg, respectively), were immobilized onto
1 mL HiTrap NHS-activated Sepharose
TM
High Perform-
ance columns (Amersham Biosciences), according to the

manufacturer’s recommendations. To provide a complex
background for affinity chromatography experiments,
100 mL of an overnight culture of E. coli,strainRR1D
M
15,
was pelleted by centrifugation, redissolved in 10 mL
1 · TST buffer and lysed by sonication. One milliliter of
the lysate into which 0.1 mg of the IgA1116 antibody had
been previously added was loaded onto the (Z
IgA1
)
2
-ABD
column. After washing with 1 · TST protein was eluted of
the column by adding 0.5
M
HAc pH 3.2. Applied sample,
flow-through and eluted proteins were analysed by SDS/
PAGE on a 10–15% PhastGel
TM
under reducing conditions
(Amersham Biosciences) and stained by Coomassie Brilliant
Blue. The eluate was also analyzed under nonreducing
conditions. One milliliter of unconditioned normal human
plasma (NHP) was applied onto the (Z
IgA1
)
2
-His
6

column at
25 °C and after washing with 10 mL phosphate buffer
Ó FEBS 2002 IgA-specific affibody ligands (Eur. J. Biochem. 269) 2649
pH 7.5 and 2 mL of 5 m
M
NH
4
Ac,pH 5.5,thecolumnwas
eluted with 0.5
M
HAc pH 3.0. Applied sample, flow-
through and eluted proteins were analysed by SDS/PAGE
on a 10–20% NovexÒ gel (Invitrogen life technologies, UK)
and stained by Coomassie Brilliant Blue. Sample, flow-
through, wash fractions and eluted proteins were further
analysed for IgA, IgG and IgM content by immunoprecip-
itation (nephlometry) at the routine laboratory at the
Karolinska Hospital, Stockholm, Sweden, using a Swedac
accredited IMMAGE instrument (Beckman Coulter, Stock-
holm, Sweden). The detection limits for IgG, IgM and IgA
assays were 0.33, 0.04 and 0.02 gÆL
)1
, respectively.
Western blotting
Sample, flow-through and eluate fractions from the affinity
chromatography experiment with unconditioned NHP were
applied on a 10–20% SDS/PAGE gel (NovexÒ) and
proteins were electroblotted to nitro-cellulose membranes
(NovexÒ). The blotted membranes were preblocked in
blocking solution (1% milk powder in TST) for 30 min at

room temperature and washed twice with TST. The
membranes were then incubated for 1 h at room tempera-
ture with horse radish peroxidase (HRP)-conjugated IgG
rabbit polyclonal antibodies directed to either human IgA,
IgG, IgM, IgD or IgE (all from Serotec, UK) diluted
1 : 1000 (IgA), 1 : 3000 (IgG), 1 : 500 (IgM), 1 : 500 (IgD)
and 1 : 500 (IgE) in 0.15
M
NaCl, 50 m
M
Tris/HCl, pH 7.5.
After washing of the membranes four times in TST for
15 min, staining was performed for  20minwitha
solution of 0.15
M
NaCl, 50 m
M
Tris/HCl, pH 7.5 supple-
mented with 0.1% H
2
O
2
and a small amount of 3,3¢-
dinitrobenzidine (Sigma–Aldrich).
RESULTS
Selection and production of affibody variants
A phage displayed protein library constructed by combina-
torial mutagenesis of surface-located positions of the IgG-
binding domain Z was used as source for the identification
of variants (affibodies) capable of selective binding to

human IgA. In order to direct the selection of IgA binding
affibody variants towards nonvariable domains, two differ-
ent human IgA
1
monoclonal antibodies (myelomas) were
used alternatively as microbead-immobilized panning tar-
gets during five cycles of affibody phage library selection. To
increase the selection stringency, increasing number of
washing steps were introduced in later cycles. DNA
sequence analyses were performed on 10 clones derived
from the fifth cycle eluate, which revealed two clones that
were represented twice (Z
IgA7
and Z
IgA4
) and an additional
six unique variants. An alignment of their amino-acid
sequences showed upon regions of similarities between some
variants (Fig. 1; see Discussion), although no obvious
overall consensus sequence could be identified. For subse-
quent binding analyses, all eight variants were produced
from their respective phagemid vectors as fusions to a
5-kDa albumin binding domain (ABD) affinity tag, facili-
tating their recovery from periplasmic fractions by HSA-
affinity chromatography. Expression levels for all eight
affibody-ABD fusions were in the 10–20 mgÆmL
)1
culture
range.
Biosensor analyses

To investigate if any of the eight candidate affibody variants
showed IgA binding, a biosensor binding study was
performed. Affibody–ABD fusion proteins (25–70 l
M
) were
separately injected over a HSA-coated sensor chip surface
for indirect immobilization via their C-terminal ABD fusion
partners, followed by an immediate injection of a solution
containing one of the IgA
1
monoclonals used as panning
target. From the resulting overlay sensorgram it can be seen
that five of the eight variants showed significant IgA
interaction, of which three variants (Z
IgA1
,Z
IgA2
and Z
IgA3
)
showed higher responses (Fig. 2). Interestingly, these vari-
ants contained identical substitutions at five of the 13
randomized positions (positions 13, 17, 27, 28 and 35) with
additional surrounding similarities, indicating a hypothet-
ical consensus motif.
Interestingly, the Z
IgA7
variant, represented twice among
the sequenced clones, did not show any IgA binding under
these conditions. The three variants showing the highest

binding responses were chosen for further binding studies in

Fig. 1. Schematic representation of the location of positions for rando-
mization and their amino-acid occupancies in selected variants. (A)
Ribbon diagram of the three-helix bundle wild type Z domain (PDB
file 1SPZ), with the 13 positions randomized during affibody library
constructions indicated (labeled green and numbers. (B) Deduced
amino-acid occupancies at the variegated 13 positions in the investi-
gated affibody variants selected for human IgA binding in this work.
+/–, confirmed IgA binding or not in biosensor studies (see text for
details).
2650 J. Ro
¨
nnmark et al. (Eur. J. Biochem. 269) Ó FEBS 2002
which their IgA binding affinities (K
d
) were determined to
0.5–3 l
M
, with the highest affinity observed for the Z
IgA1
variant which was chosen for further studies.
The binding specifity of the Z
IgA1
variant was further
analyzed in a series of biosensor binding studies. To
investigate if the IgA binding capability of the Z
IgA1
affibody could possibly be explained by any IgA binding
activity present already in the Z domain scaffold used for

library constructions, Z
wt
–ABD and Z
IgA1
–ABD fusion
proteins were analyzed in parallel as immobilized onto
sensorchip surfaces for their IgA and IgG binding capabil-
ities. The results from this analysis showed that the wild-
type Z domain did not possess any IgA binding activity,
and that the selected Z
IgA1
affibody had not retained any
detectable IgG binding ability from its ancestral Z domain
structure (Fig. 3). The isotype binding selectivity of the
Z
IgA1
affibody was further challenged with injections of
human IgM, IgD or IgE antibody preparations. No
detectable binding to these isotypes could be observed
under these conditions (Fig. 4).
In the selection of the Z
IgA1
affibody, two human IgA
1
monoclonal antibodies were used as panning targets. To
investigate if antibodies belonging to the human IgA
2
subclass also were recognized by the Z
IgA1
affibody, human

polyclonal IgA
1
and human myeloma IgA
2
samples were
analyzed for binding. The results showed that the Z
IgA1
affibody was indeed capable of binding to both IgA
1
and
IgA
2
(Fig. 4). In addition, human polyclonal IgA, contain-
ing both subclasses, was also efficiently bound by the Z
IgA1
affibody (Fig. 4). Binding was also demonstrated to secre-
tory IgA, composed of dimeric IgA linked by the J-chain
and secretory component (Fig. 4). Taken together, the
binding specificity studies suggested that the Z
IgA1
affibody
recognized an IgA-specific epitope present in all known
forms of human IgA.
Affinity recovery of IgA from a spiked
E. coli
total lysate
To investigate the potential use of the Z
IgA1
affibody as an
IgA-specific ligand in affinity chromatography applications,

an affinity column was prepared by coupling of (Z
IgA1
)
2
–
ABD fusion protein, consisting of a genetically constructed
head-to-tail dimer of the Z
IgA1
ligand fused to the serum-
albumin-binding ABD tag, to activated Sepharose using
amine coupling chemistry (see Materials and methods, and
-400
0
400
800
1200
1600
2000
2400
2800
3200
0 100 200 300 400 500 600 700 800 900 1000
Time (s)
Response units (RU)
Z
IgA1
Z
IgA2
Z
IgA3

Z
IgA4
Z
IgA5
Z
IgA8
Z
IgA7
Z
IgA6
1
2
3
Fig. 2. Overlay sensorgram from IgA binding studies of eight affibody-
ABD fusion proteins. Samples were injected over a HSA-coated surface
for an initial directed immobilization via the ABD tag–HSA inter-
action (1), directly followed by injections of a 2-l
M
solution of human
IgA
1
(myeloma) (2), ending at (3). Five variants, denoted Z
IgA1
-Z
IgA5
were confirmed to bind human IgA under these conditions.
-100
100
300
500

700
900
1100
1300
1500
1700
1900
0 200 400 600 800 1000 1200 1400
Time [s]
Response units [RU]
A
Z
wt
surface
Z
IgA1
surface
IgA injected
-100
100
300
500
700
900
1100
1300
1500
1700
1900
0 200 400 600 800 1000 1200 1400

Time [s]
Response units [RU]
Z
wt
surface
Z
IgA1
surface
IgG injected
B
Fig. 3. Initial binding specificity analysis of the Z
IgA1
variant. Resulting
overlay sensorgrams from injections of human IgA (A) or human
polyclonal IgG (B) over separate sensor chip surfaces containing either
Z
IgA1
–ABD or Z
wt
–ABD proteins, as indicated.
-100
400
900
1400
1900
2400
0 200 400 600 800 1000 1200 1400
Time [s]
Response units [RU]
pIgA

sIgA
IgA
1
IgA
2
pIgG
IgM
IgE
IgD
Fig. 4. Isotype specificity analysis of the Z
IgA1
affibody. Resulting
overlay sensorgrams from separate injections of 0.2 l
M
solutions of
human polyclonal IgA and IgA
1
,humanmyelomaIgA
2
and IgM,
human secretory IgA, human polyclonal IgG, human myeloma IgD or
recombinant mouse (light chain and VH)::human (epsilon 1–4 heavy
chain) chimera, respectively, over a sensor chip surface containing
coupled Z
IgA1
–ABD protein.
Ó FEBS 2002 IgA-specific affibody ligands (Eur. J. Biochem. 269) 2651
Discussion). This dimerization did not change the isotype
binding specificity of the IgA affibody ligand, as determined
by biosensor technology (data not shown). To provide a

complex background of proteins for testing of the selectivity
of the ligand, monoclonal IgA protein was spiked
(0.1 mgÆmL
)1
) into an E. coli total lysate obtained by
sonication of a stationary phase bacterial culture. After
sample loading and subsequent washing, the column was
eluted with low pH. Analysis by SDS/PAGE (reducing
conditions) of the flow-through and eluate fractions showed
that the IgA material had been efficiently recovered with
high purity (Fig. 5). Analysis by nonreducing SDS/PAGE
showed that the eluate contained a band of high molecular
mass but no material migrating as separate heavy and light
chains, indicating that intact IgA had been recovered (data
not shown). This showed that the micromolar affinity of the
interaction was sufficient for affinity chromatography and
that the selectivity of the ligand was high.
Affinity recovery of IgA from human plasma
The potential use of the Z
IgA1
affibody as affinity ligand for
selective chromatographic recovery of IgA directly from
human blood fractions was also investigated. In this
experiment unconditioned normal human plasma was used
as the source of IgA, containing in addition to IgA also all
other antibody isotypes and a highly complex mixture of
other proteins [30]. For this experiment, due to the presence
of serum albumin in plasma, an affinity column was
prepared through coupling of an alternative dimeric ligand
devoid of the albumin binding ABD tag [denoted (Z

IgA1
)
2
-
His
6
] to activated Sepharose media (see Materials and
methods) and coupled to a chromatographic system. One
milliliter of human plasma sample was applied and samples
from the flow through, wash and eluate (low pH) fractions
were collected for later analyses. A chromatogram (A
280
)
from a typical experiment is shown in Fig. 6, with a large
flow-through peak, followed by base-line values during
washing and a significant eluate peak. Nephlometry analy-
ses for IgG, IgM and IgA in the different fractions showed
that IgG and IgM were present in the flow-through and
early wash fractions, but not in the eluate. In contrast, IgA
could not be detected in the flow-through but was present at
high concentration in the eluate, indicating an efficient and
selective capture of IgA.
An SDS/PAGE analysis of the sample, flow-through and
eluate fractions showed that the recovered IgA had been
efficiently purified (Fig. 7A). For a more sensitive analysis
than provided by the nephlometry method, the efficiency of
the procedure and the purity of the recovered IgA was
further tested by immunoblotting on the same fractions with
reagents specific for human IgA, IgG, IgM and IgD isotypes
(Fig. 7B–E). As expected, all four isotypes could be detected

in the applied sample. For the flow-through fractions,
similar staining intensities as in the sample fractions were
observed for IgG, IgM and IgD, indicating that these
isotypes were not significantly retarted in the column.
Interestingly, IgA could not be detected in the flow-through
fraction, supporting the notion that this isotype had been
efficiently recovered from the plasma sample. The analyses
of the eluate fraction showed upon a major IgA content as
expected, but with small amounts of contaminating IgG and
IgM (see below).
DISCUSSION
In this work, a combinatorial protein engineering approach
was used to obtain novel IgA binding proteins. Using the
well-characterized protein A-derived IgG-binding, three-
helix bundle domain Z as scaffold for library constructions,
Fig. 5. SDS/PAGE analysis (reduced conditions, Coomassie Brilliant
Blue staining) of different samples from the recovery of IgA protein from
aspikedE. coli total lysate. Lane 1, applied sample; lane 2, flow-
through fraction and lane 3, eluate fraction. The reducing conditions
results in the separation of the IgA into heavy and light chain frag-
ments.
-200
0
200
400
600
800
1000
1200
1400

1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
4200
0123456789101112131415161718
Fraction
A
280
[mAU]
Acetic acidPBSSample
NH
4
Ac
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Fig. 6. Chromatogram from the affinity chromatography purification of
IgA from unconditioned normal human plasma using an affinity column
containing a divalent (Z
IgA1
)
2

-His
6
ligand. Intervals for sample loading,
NaCl/P
i
buffer washing, ammonium actetate buffer washing and low
pH elution are indicated. The A
280
was monitored throughout the
experiment.
2652 J. Ro
¨
nnmark et al. (Eur. J. Biochem. 269) Ó FEBS 2002
five out of eight investigated variants selected using phage
display technology showed IgA binding. The reasons
leading to the selection of non-IgA binding variants were
not investigated further, but could be due to background
binding of such variants to components other than IgA
present during selection, such as streptavidin beads and
phages. In several of the confirmed binders, some substitu-
tions were identical with a preference to the second
variegated helix (Fig. 1). This could indicate that these
variants binds to a common site on the IgA protein.
One of the confirmed IgA-binders (clone Z
IgA1
) was
characterized further and showed to be selective for human
IgA in a series of biosensor binding studies, where the
specificity was challenged with all other human isotypes,
including polyclonal IgG. Further studies showed that both

IgA
1
and IgA
2
subclasses, as well as secretory IgA, was
efficiently recognized by the Z
IgA1
variant. In comparison to
three described natural bacterial IgA binding proteins, this
gives the Z
IgA1
affibody an IgA binding profile closer to
those of the Arp4 and Sir22 proteins from Streptococcus
pyogenes than that of the serologically distinct IgA-binding
receptins from group B streptococci, showing only weak
binding to secretory IgA [31]. However, no obvious
sequence similarity can be seen between the Z
IgA1
affibody
sequence and a 29-residue motif that shows sequence
similarity between the Arp4 and Sir22 proteins and has been
postulated to contain the IgA binding regions in these
proteins. Interestingly, a 50-residue peptide derived from
Sir22 containing this motif as central element was found to
bind IgA with high specificity [18], but was described to be
dependent on the formation of coiled-coiled dimers for IgA
binding.
The binding sites for the three natural streptococcal IgA
binding receptins were recently mapped to the CH
2

–CH
3
region interface of the Fc fragment, overlapping with the
binding site for the human CD89 IgA receptor [19],
suggesting biological significance. It is worth noting that
in the interactions between IgG and protein A or protein G,
the corresponding domain interface is involved in the
binding [32]. However, the IgA binding proteins described
in this work were selected in vitro, in the absence of any
obvious biological selection pressure, suggesting that more
or less any site on the IgA heavy chain not protected by the
secretory component could contain the binding epitope. The
recognition of both the IgA
1
and IgA
2
subclasses suggests
that the IgA hinge region, which differs in length and
sequence between the two forms of IgA, is not significantly
involved in the binding.
In the affinity chromatography experiments, head-to-tail
dimeric constructs of the Z
IgA1
affibody were used, resulting
in divalent ligands. Although the primary reason for the
dimerization was to increase the likelihood that at least one
moiety in each dimeric ligand was biologically active after
the amine coupling procedure, a divalent capturing ligand
could potentially also result in adventageous avidity effects
in the binding between the ligand and the target as well as to

provide a beneficial effect on the presentation of the
immobilized ligand to the surrounding solution related to
the use of a larger ligand (spacer effect).
The efficient recovery of IgA from the unconditioned
human plasma suggests that the described ligand could be of
interest to evaluate for use in extracorporeal IgA-removal
applications, of potential relevance for investigation and
treatment of IgA nephropathy. Interestingly, whereas the
biosensor binding data showed that the Z
IgA1
affibody was
highly selective for IgA, small amounts of IgM and IgG
could be detected by Western blotting in the eluate from the
recovery of IgA from human plasma. This could possibly be
explained by the presence of natural immunoglobulin-
specific autoantibodies in healthy individuals, resulting in
the formation of circulating immune complexes (CIC). Such
natural autoantibodies have been described to be of IgM,
IgG and IgA isotypes [33]. In addition, anti-(protein A) Ig,
recognizing the nonvariegated parts of protein A-derived
affibody ligand structure, could also be present in the
plasma, as a result of previous bacterial infections of the
plasma donors.
A growing number of antibodies intended for thera-
peutic use are currently under investigation in clinical
trials [34]. The majority of those are of IgG isotype, which
can effectively activate complement- and antibody-
dependent cellular cytotoxicity pathways. In addition,
Fig. 7. SDS/PAGE (reduced conditions, Coomassie Brilliant Blue staining) of sample (S), flow-through (FT) and eluate (E) samples from the affinity
chromatography experiment shown in Fig. 6 (A), and corresponding human isotype-specific Western blotting analyses of the same samples (B–E). For

the isotype analysis different human isotype-specific horse radish-conjugated rabbit IgG sera were used as follows: anti-(human IgA) Ig (B), anti-
(human IgG) Ig (C), anti-(human IgM) (D) and anti-(human IgD) Ig (E).
Ó FEBS 2002 IgA-specific affibody ligands (Eur. J. Biochem. 269) 2653
antibodies of the IgG isotype are effectively purified by
protein A affinity chromatography, which is also imple-
mented at industrial scale processes [35]. However, the
IgA isotype has also been suggested for immunotherapy
applications, owing its strong association with neutrophils
and the possibility to direct antibodies to luminal surfaces
[17]. For example, Streptococcus mutans-specific IgA has
been investigated for administration to the oral cavity for
the prevention of caries [36]. In analogy to the efficient
protein A-based purification of IgG, the development of
corresponding affinity chromatography media containing
a robust affinity ligand selective for IgA should facilitate
the purification of recombinant antibodies of this isotype.
Based on the stable structure of a single protein A
domain, the ligands of the type described in this work
could constitute interesting candidates for such applica-
tions.
ACKNOWLEDGEMENTS
This work has been supported by support from the Swedish Research
Council for Engineering Sciences (TFR), the Swedish Natural Science
Research Council (NFR) and Amersham Biosciences.
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