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Clinical Chemistry 48:12
2208 –2216 (2002)
Proteomics and
Protein Markers
Epitope Mapping of Antibodies against Prostatespecific Antigen with Use of Peptide Libraries
Jari Leinonen,* Ping Wu, and Ulf-Håkan Stenman
Background: Prostate-specific antigen (PSA) is the most
important marker for prostate cancer, but PSA concentrations determined by various assays can differ significantly because of differences in specificity of the antibodies used. To identify epitopes recognized by various
monoclonal antibodies (MAbs) to PSA, we have isolated
peptides that react with the paratopes of these.
Methods: Six anti-PSA MAbs representing three major
epitope groups were screened with five cyclic phage
display peptide libraries. After selection, the peptide
sequences were determined by sequencing of the relevant part of viral DNA. Binding of the phage peptides to
the MAbs was monitored by immunoassay.
Results: For each MAb, several paratope-binding peptides with distinct sequence motifs were identified, but
only ⬃10% showed similarity with the PSA sequence.
Some of these correctly predicted the location of the
epitopes. By sequential panning of the library with two
closely related MAbs, we identified peptides reacting
equally with both MAbs. When analyzed against a large
panel of PSA MAbs, the peptides generally showed
restricted specificity toward the MAb used for selection,
but some peptides bound to several related MAbs.
Conclusions: Most of the cyclic peptides selected with
PSA MAbs are specific for the MAb used for selection
and do not resemble any sequence on the antigen.
Peptides reactive with two MAbs recognizing the same
epitope can be obtained by sequential panning. This
method can be used to predict the location of some
epitopes, but additional methods are needed to confirm
the result.
© 2002 American Association for Clinical Chemistry
Prostate cancer is the most common malignancy of males
and the second leading cause of cancer death in the US
Department of Clinical Chemistry, Helsinki University Central Hospital,
FIN-0029 Helsinki, Finland.
*Address correspondence to this author at: Department of Clinical Chemistry in Biomedicum, Helsinki University Central Hospital, Haartmaninkatu 8,
FIN-00290 Helsinki, Finland. Fax 358-9-4717-1731; e-mail [email protected].
Received May 27, 2002; accepted August 30, 2002.
(1 ). Prostate-specific antigen (PSA)1 is a serine protease
secreted by the epithelial cells of the prostate (2 ). It is a
reliable marker for monitoring treatment of prostate cancer, but it is also widely used for early diagnosis and
screening (3, 4 ). Serum PSA is frequently increased in
patients with benign prostatic hyperplasia, and in a
screening setting, only approximately one-third of those
with increased PSA have prostate cancer (4 ). PSA exists in
circulation in various molecular forms, including several
PSA-inhibitor complexes and isoforms of free PSA, and
the use of assays for these forms can improve the clinical
accuracy of PSA determination (5–9 ). The PSA concentrations measured by different assays vary significantly,
which can be attributable to differences in standardization
or specificity of the antibodies used toward various molecular forms of PSA (10 ). Furthermore, some PSA antibodies can also recognize human kallikrein 2 (hK2), which
may affect the measured PSA concentrations. hK2 shows
⬃80% identity with PSA at the amino acid level (11–13 ),
and like PSA, hK2 is a prostate-derived proteinase that
has been shown to be a useful marker for prostatic disease
(14, 15 ). PSA has been shown to contain six major epitope
regions, which are exposed to a varying extent in different
forms of PSA (16 ). To develop assays with highly defined
specificity, it is important to know the epitopes of the
antibodies used in the assay. Use of antibodies with
defined specificities facilitates the development of assays
that produce comparable results and the design of assays
specific for variant forms of the antigen. Although the
epitopes of PSA have been studied extensively (16 –22 ),
the locations of only a few of the epitopes have been
localized on the primary sequence of PSA. Amino acid
residues forming linear epitopes can be identified by
synthetic peptide epitope mapping, in which short chemically synthesized peptides covering the whole coding
sequence of the antigen are used to identify the regions
interacting with the monoclonal antibodies (MAbs) (23 ),
1
Nonstandard abbreviations: PSA, prostate-specific antigen; hK2, human
kallikrein-2; MAb, monoclonal antibody; IFMA, immunofluorometric assay;
and TBS, Tris-buffered saline.
2208
Clinical Chemistry 48, No. 12, 2002
as also described for PSA (19, 20 ). An alternative method
is to screen peptide libraries to identify paratope-binding
peptides, i.e., peptides binding to the antigen-binding site,
and to compare the structures of these with that of the
antigen (21, 24 –26 ). We studied whether paratope-binding peptides to MAbs against PSA isolated by screening
cyclic phage display peptide libraries can be used to
predict the structure of PSA epitopes.
Materials and Methods
antibodies and proteins
The MAbs 5E4, 9C5, and 4G10 were developed with the
following protocol. BALB/c mice were immunized with
10 –30 ␮g of purified PSA by intraperitoneal injection with
Freund’s complete adjuvant. PSA was purified from human seminal fluid as described previously (27 ). A booster
dose of 10 ␮g was given after 4 weeks, and then additional
boosters of 100 and 150 ␮g were given 1 and 2 days after
the first booster, respectively. After the final booster, the
splenic lymphoid cells of the mice were fused with the
mouse myeloma cell line P3x63-Ag8.653 (American Type
Culture Collection). The fused cells were harvested in
HAT medium supplemented with interleukin-6 for 4
weeks, after which the cells were harvested in HT medium. PSA antibody production was screened by use of
an immunofluorometric assay (IFMA) in microtiter wells
coated with rabbit anti-mouse immunoglobulin. Briefly,
25 ␮L of hybridoma supernatants plus 200 ␮L of IFMA
assay buffer (6 ) were incubated for 1 h in the wells, after
which the wells were washed twice and 10 ng of Eu3⫹labeled PSA in 200 ␮L of assay buffer was added. After
additional incubation for 30 min, the wells were washed
four times, and 200 ␮L of enhancement solution (PerkinElmer-Wallac) was added. After incubation for 5 min, the
fluorescence was measured. The MAbs produced were
purified by affinity chromatography using protein G
columns (Amersham Biosciences).
Anti-PSA MAbs H117 and H50 were obtained from
Abbott Diagnostics. Antibody 5A10 was from PerkinElmer-Wallac. The characteristics of the ISOBM workshop
panel of anti-PSA MAbs have been described in detail
elsewhere (16 ). Anti-phage MAb was a kind gift from Dr.
Petri Saviranta (University of Turku, Turku, Finland).
Anti-phage polyclonal antibody was purchased from Amersham Biosciences. Peptides were synthesized by standard Merrifield solid-phase chemistry. The phage peptide
libraries were a kind gift from Dr Erkki Koivunen (University of Helsinki, Helsinki, Finland).
selection of phage peptides
Screening of the phage display peptide libraries was
performed essentially as described previously (28 ). Libraries with the structures CX7C, CX8C, CX10C,
CX3CX3CX3C, and CX3CX4CX2C, where C is cysteine
and X is any of the 20 naturally occurring amino acids,
were used. A pool of these libraries containing 1011–1012
infectious particles was screened with each MAb. Briefly,
2209
a pool of phage libraries was added to the wells coated
with a MAb and incubated for 3 h at 22 °C during the first
round of panning and for 1 h during subsequent rounds.
The phage solution was removed, and the wells were
washed with Tris-buffered saline (TBS; 50 mmol/L TrisHCl, pH 7.8, 0.15 mol/L NaCl) containing 5 mL/L Tween
20. Bound phage were eluted with 0.1 mol/L glycine
buffer, pH 2.2, and neutralized with 1 mol/L Tris base.
The eluted phage were amplified by infection of Escherichia coli K91 kan cells and purified by precipitation with
polyethylene glycol. Sequential panning with two MAbs
was performed with MAb pairs known to react with the
same antigenic region on PSA, including MAb pairs
5E4/H117, 9C5/H50, and 4G10/5A10. For example, the
primary library was panned for two rounds using MAb
5E4, after which the eluate was amplified and panned for
one round with MAb H117.
After three rounds of selection and amplification, the
peptide sequences were determined by sequencing the
relevant part of the viral DNA as described previously
(28 ). Sequencing was performed with an ABI 310 Genetic
Analyzer and Dye Terminator Cycle Sequencing Core Kit
(PE Applied Biosystems), with the oligonucleotide 5⬘CCCTCATAGTTAAGCGTAACG-3⬘ as a primer. The
amino acid sequence of each peptide isolated was analyzed for homology with PSA and other proteins by the
BLASTP program using the SwissProt database. The
three-dimensional model of PSA established by
Villoutreix et al. (29 ) was visualized with the RasMol
program (30 ).
IFMAs
The solid-phase antibodies used in the IFMA were coated
on microtitration wells at a concentration of 5 mg/L in
TBS for 16 h at 22 °C. The solution was then discarded,
and the wells were saturated with 10 g/L bovine serum
albumin in TBS for 3 h at 22 °C. The antibodies used as
tracers were labeled with a Eu3⫹ chelate as described
previously (28 ). The assay buffer was TBS, pH 7.7, containing 33 ␮mol/L bovine serum albumin and 1 ␮mol/L
bovine globulin.
The binding of individual phage clones to MAbs was
tested by phage IFMA. In this assay, 15 ␮L of phage
(109–1010 infectious particles) and 200 ␮L of assay buffer
were added to wells coated with each MAb. After incubation for 1 h, the wells were washed and filled with 200
␮L of assay buffer containing 50 ng of a Eu3⫹-labeled
anti-phage MAb recognizing the M13 coat protein of the
phage. After incubation for 60 min, the wells were washed
four times, and enhancement solution (Perkin-ElmerWallac) was added. Fluorescence was quantified with a
1234 DELFIA Research fluorometer (Perkin-Elmer-Wallac). The detection limit was defined as the fluorescence
signal obtained with phage containing unrelated peptide
⫹3 SD.
The binding specificity of the phage peptide to the
antigen-binding regions of the mAbs was confirmed by
2210
Leinonen et al.: Epitope Mapping of PSA by Peptide Libraries
showing that PSA inhibited the binding of phage to
antibodies. Briefly, 15 ␮L of each phage clone and 0.3–300
ng of PSA purified from seminal fluid were pipetted into
a well coated with each mAb. After incubation of phage
and PSA for 2 h, the protocol for phage IFMA was
followed. The binding specificity of synthetic peptides
was confirmed by showing that they competed with PSA
for binding to antibodies. Briefly, 0 –120 ␮g of peptide in
200 ␮L of assay buffer was pipetted into a well coated
with MAb. After incubation for 1 h, 5 ng of PSA was
added and incubated for 30 min. Inhibition of PSA
binding was quantified by PSA IFMA as described (31 ).
reactivity of the phage with the isobm workshop
panel of MAbs
Phage isolated by panning with the six PSA MAbs were
tested for binding to 39 MAbs from the ISOBM workshop
for characterization of antibodies against PSA by phage
IFMA. According to the ISOBM classification, the six
MAbs used for screening of the phage libraries belonged
to epitope groups 1, 3, and 6 (16 ), which in this study are
referred to as I, III, and VI, respectively. The ISOBM
workshop MAbs tested included MAbs 26, 33, 68, 74, 77,
78, 85, 209, 213, 216, 223, and 230 from epitope group I;
MAbs 31, 36, 57, 63, 64, 66, 72, 82, 84, 88, 89, 212, 224, and
229 from epitope group III; and MAbs 27, 29, 34, 56, 65, 67,
75, 79, 210, 214, 218, 221, and 225 from epitope group VI.
Results
identification of antibody-binding peptides
The MAbs studied were chosen on the basis of crossinhibition characteristics to include three pairs of closely
related antibodies. The peptides isolated with the six
MAbs contained characteristic motifs and consensus
amino acids (Table 1), but only 9 of 100 peptides displayed sequence identity with PSA, defined as at least
three identical amino acids within a window of four
amino acids. Binding of the peptides to MAbs was inhibited by PSA, showing that they were paratope specific
(Fig. 1).
The epitopes of MAbs 5E4 and H117 have been
mapped to the N-terminal region of PSA (amino acid
residues 3–11) by synthetic peptide mapping and crossinhibition studies (16, 20 ). However, most of the peptides
developed against these antibodies resembled neither
PSA nor each other. This indicated that the peptides
recognized structures in the paratope that do not participate in antigen binding or that the epitopes were different
and mainly determined by the three-dimensional structure of PSA. To identify peptides reacting with two MAbs
recognizing the same epitope, we developed a sequential
panning strategy. The peptides identified by this strategy
using MAbs 5E4 and H117 did not resemble the peptides
selected separately (Table 1). Two peptides, CPSVDGGWTC (VI-13 and VI-20) and CHSACSKHCFVHC (VI-15
and VI-22), were obtained irrespective of the order in
which the MAbs were used for selection. In peptides
Table 1. Amino acid sequences of peptides selected from
phage display peptide libraries with MAbs against PSA.a
MAb(s)
Peptide
Sequence
5E4
5E4
5E4
5E4 and H117
5E4 and H117
5E4 and H117
H117
H117 and 5E4
H117 and 5E4
H117 and 5E4
5A10 and 4G10
5A10 and 4G10
5A10 and 4G10
5A10 and 4G10
4G10
4G10 and 5A10
4G10 and 5A10
4G10 and 5A10
4G10 and 5A10
4G10 and 5A10
H50
H50
H50
H50
H50 and 9C5
H50 and 9C5
H50 and 9C5
H50 and 9C5
H50 and 9C5
9C5
9C5 and H50
9C5 and H50
9C5 and H50
VI-1
VI-9
VI-12
VI-13
VI-14
VI-15
VI-18
VI-20
VI-21
VI-22
I-8
I-9
I-15
I-16
I-19
I-30
I-31
I-32
I-33
I-35
III-1
III-8
III-9
III-12
III-14
III-15
III-17
III-20
III-25
III-28
III-37
III-38
III-39
CPVDFDFLC
CPRDFEFLC
CPADFEFLC
CPSVDGGWTC
CKSMDGGWTC
CHSACSKHCFVHC
CTWHWSPEEC
CPSVDGGWTC
CHSACSKHCFVYC
CHSACSKHCFVHC
CRPWGSSRC
CKSWGSSRC
CNLYKVGC
CHPYKVGC
CSVYPFWHC
CRSYPFWMC
CVSYPFWKC
CAVFPFWRC
CVWWWGC
CAPWWGC
CSGIAPWLC
CGPGIDSWVC
CGPGIRSWVC
CDYMPLVDNC
CEGIAVWLC
CTRYSGYWVC
CTQMNGYWIC
CLYDHLC
CILLYNDCC
CFFGNWNFC
CWNWNLHISC
CYFKNWNFC
CVRSCISDCELRC
a
The sequences of the peptides analyzed further are shown. The peptides
were isolated by panning with each MAb or by sequential panning, in which the
library was first panned using one MAb, after which the eluate was panned with
another related MAb. The peptide sequences were determined by sequencing
the relevant part of the viral DNA. MAbs 5E4 and H117 belong to epitope group
VI, MAbs 5A10 and 4G10 to group I, and MAbs H50 and 9C5 to group III (16 ).
The sequences of all peptides isolated (n ⫽ 100) are available as a data
supplement with the online version of this article at http://www.clinchem.org/
content/vol48/issue12/.
VI-13 and VI-14, the motif GGWTC was similar to the
sequence GGWEC comprising residues 3–7 in the NH2
terminus of mature PSA (Fig. 2). Furthermore, in peptides
VI-15 and VI-21, the motif CSKH was similar to the
sequence CEKH comprising residues 7–10 in PSA (Fig. 2).
Thus, the structures of these peptides suggest that the
epitope recognized by MAbs 5E4 and H117 comprises
residues 3–10 in mature PSA. All phage peptides isolated
by use of MAb 5E4 alone contained the structure PXDFXFL. This motif was not present in peptides selected by
MAb H117 alone, which contained the motif WXXXPXE
(Table 1). Among MAb 5E4-selected peptides, peptides
Clinical Chemistry 48, No. 12, 2002
Fig. 1. Inhibition of phage binding to anti-PSA MAb by PSA.
We incubated 15 ␮L of each phage clone (1011–1012 infectious particles) with
increasing concentrations of PSA (0 – 400 nmol/L) in a well coated with MAb
H117. After washing, the binding of phage was monitored by an Eu3⫹-labeled
anti-phage antibody. The reactivity is shown for phage clones VI-13 (E), -14 (䡺),
-15 (Œ), and -21 (‚) obtained by sequential panning using MAbs 5E4 and H117.
The background of ⬃1000 cps has been subtracted.
VI-9 and VI-12 contained the sequence EFL corresponding
to PSA amino acid residues 140 –142; a similar sequence,
DFL, was present in four other peptides, and the FL
sequence was present in all 12 peptides. A synthetic
peptide (corresponding to the sequence of phage peptide
2211
VI-12) containing the EFL sequence inhibited the binding
of PSA with MAb 5E4 in a dose-dependent manner (Fig.
3), confirming that this peptide and PSA bind to same site
on the MAb. Among MAb H117-selected peptides, peptide VI-18 contained the PEE sequence (residues 138 –140
in PSA), and the PXE motif was present in three of the
four peptides. Thus, these MAbs recognize the same
amino acids in region 3–10 and different amino acids from
the region 138 –142 (PEEFL; Fig. 2). Interestingly, in the
three-dimensional model of PSA (29 ), the PEEFL sequence is located close to the N-terminal epitope (Fig. 4).
These results suggest that amino acids 138 –142 form part
of a noncontinuous epitope together with amino acids
3–10 in the N-terminal region.
Group I MAbs 5A10 and 4G10 bind to an epitope
specific for free PSA, which in PSA-serpin complexes is
covered by the inhibitors (16, 32 ). Some MAbs in this
group have been shown to bind to linear epitopes comprising amino acids 80 –91 in PSA, which form part of the
so-called kallikrein loop (16, 20 ), but most of them react
only weakly with reduced, intact PSA, suggesting that
they are mainly conformation dependent. MAbs 5A10
and 4G10 inhibit each other efficiently, but MAb 4G10 has
been shown to cross-react with complexed PSA approximately two- to fivefold more than does 5A10 (33, 34 ),
suggesting that the epitopes of these MAbs are not
identical. When we selected with 4G10 and sequentially
with 4G10 and 5A10, we found the motifs YPFW, WWW,
and PWW (Table 1), but these are not found in PSA.
Fig. 2. Amino acid sequence and epitope structure of PSA.
Residue 1 indicates the N-terminal residue of the active form of PSA. The sequences and codes of the peptides used to assign the epitopes are shown above the PSA
sequence with the identical residues underlined. Previously determined locations for the epitope groups of MAbs included in the present study are as follows: group
VI, amino acid residues 3–11; group I, residues 80 –91 (16, 20 ), 145–148, and 204 –208 (21 ); group III, residues 151–155 (20 ). Active site residues His-41, Asp-96,
and Ser-189 are underlined.
2212
Leinonen et al.: Epitope Mapping of PSA by Peptide Libraries
Fig. 3. Inhibition of PSA binding to MAb 5E4 by a synthetic peptide.
We incubated 0 – 600 mg/L of chemically synthesized peptide CPADFEFLC (VI-12
in Table 1) in a well coated with MAb 5E4 for 30 min, after which 0.4 ng of PSA
was added and incubation was continued for an adiditional 30 min. After
washing, the inhibition of PSA binding was quantified by PSA IFMA (31 ). The
columns represent mean values of duplicate measurements ⫾ SD (error bars).
However, when we selected with MAb 5A10 and sequentially with 5A10 and 4G10, we frequently found the WGS
and TVXXAW motifs. Peptide I-9 contained the sequence
SWGSSRC, which is similar to the PSA sequence SWGSEPC (amino acids 204 –210 in PSA; Fig. 2). Furthermore,
WGS, WG, or GS motifs were present in 7 of 22 peptides
isolated. In agreement with this, Michel et al. (21 ) have
found by screening peptide libraries that the SWG site
forms part of the epitope of MAbs specific for free PSA.
Amino acids 204 –207 (SWGS) form the edge of the groove
containing the active site residues of PSA (29 ) opposite
the region 80 –91 on the other side of the groove. Peptide
I-16 contained the sequence HPYKV, which is similar to
the PSA sequence HPQKV (amino acids 164 –168 in PSA),
which is located adjacent to amino acids 204 –207 and
borders to the predicted binding site of group III MAbs
(Fig. 2). It may thus form part of the epitope for MAbs
specific for free PSA. Further support for the location of
the epitope as predicted by phage display peptides can be
obtained by analyzing whether the predicted epitope
regions differ between PSA and hK2 and accounting for
whether the MAb reacts with PSA only or with both PSA
and hK2. hK2 shares 80% identity with PSA (11–13 ), and
many PSA antibodies are known to recognize hK2, showing that several epitopes in PSA and hK2 are identical
(16, 35 ). Thus, the location of the epitope of MAb 5A10 in
the 164 –168 region in PSA is further supported by the
facts that this region differs between PSA and hK2
(HPQKV and YSEKV, respectively) and that MAb 5A10
Fig. 4. Molecular model of PSA (29 ) showing with “spacefill” the
epitopes for MAbs belonging to groups I and VI as identified by the
paratope-binding peptides.
The dark gray residues constitute amino acids 3–10 (GGWECEKH), similar to
peptides VI-13, -14, -15, and -21, and amino acids 138 –142 (PEEFL), similar to
peptides VI-12 and VI-18 obtained by panning with group VI MAbs. The white
residues constitute amino acids 164 –168 (HPQKV), similar to peptide I-16, and
amino acids 204 –207 (SWGS), similar to peptide I-9 obtained by panning with
group I MAbs. In addition, amino acids 81–90, previously identified as the
binding site for group I MAbs (20 ), are shown in white, and the active site amino
acids His-41, Asp-96, and Ser-189 are indicated with dots.
does not react with hK2 (16 ). Thus, these results suggest
that the epitopes for MAbs belonging to group I may
comprise residues on three different loops, i.e., amino
acids 80 –91 (20 ), 164 –168, and 204 –207.
MAbs H50 and 9C5 bind to epitopes located remote
from the active site and are considered to be mainly
conformation dependent (16 ). These MAbs show highly
similar binding characteristics, but only H50 shows strong
reactivity with hK2 (16, 36 ). The peptides isolated by
panning with MAb H50 and sequentially with MAbs H50
and 9C5 frequently contained the structures GIXXW or
GYW, whereas in peptides isolated by use of MAb 9C5
and sequentially with MAbs 9C5 and H50, the NWN
motif was found repeatedly. Peptides III-8 and III-9 (H50
selection) contained the sequences PGIDS and PGIRS,
respectively, which are similar to the sequence PGDDS in
PSA (amino acids 89 –93; Fig. 2). This region partially
overlaps amino acids 80 –91 in PSA, which is the previously identified binding site for MAbs specific for free
PSA (20 ). Furthermore, MAb H50 binds strongly to both
PSA and hK2, but the amino acid sequence 89 –93 in these
differs (PGDDS and PDEDS in PSA and hK2, respectively). Thus, it is unlikely that the PGDDS sequence is part of
the epitope for these MAbs. In peptide III-12 (H50 selection), the sequence PLVDN is similar to the PSA sequence
2213
Clinical Chemistry 48, No. 12, 2002
PLVCN (amino acids 192–196 in PSA; Fig. 2). This sequence is identical in PSA and hK2, and it is adjacent to
epitope region 151–155 in the three-dimensional model of
PSA, which previously has been predicted to be a binding
site for MAbs belonging to this epitope group. Thus, these
results suggest that the epitopes for group III MAbs
comprise at least two different loops, i.e., amino acids
151–155 (20 ) and 192–196.
reactivity of the peptides with isobm MAbs
To study whether the epitopes found were recognized by
other MAbs, we studied the reactivity of 19 peptides with
a panel of MAbs from the ISOBM workshop. The peptides
studied were isolated by sequential panning using MAb
pairs 5E4/H117 (group VI), 4G10/5A10 (group I), and
H50/9C5 (group III).
The reactivity of three group VI peptides showing
similarity with amino acids 3–10 in PSA was studied with
13 group VI MAbs (Table 2). Seven MAbs bound to these
peptides, suggesting that amino acids 3–10 in PSA form
part of the epitope recognized by most MAbs in this
group. Six of the MAbs did not bind any of these peptides,
suggesting that these amino acids are not a major part of
their epitopes or that the motif is not present in correct
conformation for these MAbs.
The reactivity of six group I peptides (I-8, I-15, I-30,
I-31, I-32, and I-35) was studied with 14 group I MAbs.
These peptides showed highly restricted specificity and
reacted only with the primary selection MAb, i.e., peptides I-8 and I-15 reacted only with MAb 5A10, and
Table 2. Reactivity of group VI MAbs with peptides isolated
by panning sequentially with MAbs 5E4 and H117.a
Peptide
MAb
VI-13
VI-14
VI-15
PSA-Eu3ⴙ
27
29
34
56 (H117)
65
67
75
79 (5E4)
210
214
218
221
225
9831
⫺
476784
472197
⫺
20575
⫺
337648
⫺
29145
⫺
⫺
1266
⫺
⫺
⫺
485780
⫺
⫺
⫺
246281
⫺
1391404
⫺
⫺
⫺
12730
⫺
14329
396210
⫺
65068
⫺
217960
⫺
53310
⫺
⫺
2072
32 410
209 062
310 266
330 600
303 059
55 636
37 636
378 440
348 434
389 005
327 034
259 856
377 816
a
The binding of phage was measured by IFMA using each of the PSA MAbs on
solid phase and anti-phage antibody as tracer. The results are expressed in
europium fluorescence units (cps) and represent mean values of duplicate
measurements. For comparison, the response (cps) obtained by adding 10 ng of
europium-labeled PSA to wells coated with each MAb is shown. A result is
indicated by (⫺) when the response was below the detection limit of the assay
(1125 cps; defined as binding of phage containing an unrelated peptide to MAbs
⫹3 SD). The assay CVs of phage binding IFMAs were 5–20%.
peptides I-30, I-31, I-32, and I-35 reacted only with MAb
4G10.
The reactivity of eight group III peptides was tested
with 14 MAbs from epitope group III (Table 3). Some of
the peptides reacted with several MAbs within this
epitope group. Most of them showed unique binding
patterns, but MAbs 72 and 229 may have closely related
specificity because they reacted only with peptide III-15.
MAb 224 reacted only with peptides III-15, -20, and -25,
obtained with MAb H50 as primary selection antibody,
whereas MAb 89 reacted only with peptide III-37, obtained with MAb 9C5 as primary selection MAb. Four
MAbs did not bind to any of the peptides.
Discussion
The main antigenic regions of PSA recognized by mouse
antibodies have been studied extensively (16, 17, 20, 22 ),
but not much is known about the fine structure of the
epitopes. In this study, we isolated peptides binding
specifically with the paratope regions of six PSA MAbs
and studied their reactivity with a large panel of PSA
antibodies.
Alignment of the peptides with the PSA sequence
revealed several binding motifs, but most of the isolated
peptides did not resemble any part of PSA. These may
represent mimotopes, i.e., antigenic mimics, or they may
be structurally unrelated to PSA, reflecting the polyspecific nature of the binding region of an antibody (37 ).
Thus, although MAbs are usually considered to be specific for a certain antigen, the paratope can also bind
structures that do not resemble the original immunogen.
Even if MAbs have rarely been shown to bind strongly to
unrelated natural antigens, cross-reactivity with a totally
unrelated protein has been observed with an antibody
prepared against a synthetic peptide (38 ). The polyspecificity can be explained by the fact that the paratope
consists of ⬃20 amino acids, but only a few of these
participate in antigen binding, and water molecules can
fill the remaining space between the paratope and
epitope. Thus, the same paratope can contain several
subsites for different epitopes, and amino acids in the
paratope can form strong bonds with peptides unrelated
to the immunogen (37, 39 ). The polyspecific nature of
paratopes toward unrelated peptides has also been demonstrated by Keitel et al. (40 ) and Kramer et al. (41 ), who
used x-ray structural data of three peptides complexed
with an anti-p24 (HIV-I) MAb.
In our study, only ⬃10% of the peptides showed
similarity with PSA. Some of these corresponded to
earlier identified epitopes, whereas others contained new
motifs adjacent to earlier described ones. Thus, they may
well form part of the epitope. Antibodies 5E4 and H117
identified the same N-terminal epitope (amino acids 3–10)
as was found in previous studies for MAbs in this group
(20 ), but they also appeared to react with amino acids in
the 138 –142 region. These antibodies also react with hK2
(16 ), and regions 3–10 and 138 –142 are identical in PSA
2214
Leinonen et al.: Epitope Mapping of PSA by Peptide Libraries
Table 3. Reactivity of group III MAbs with peptides isolated by panning sequentially with MAbs H50 and 9C5.a
Peptide
MAb
31
36
57 (H50)
63
64
66
72
82 (9C5)
84
88
89
212
224
229
a
III-14
III-15
III-17
4571
⫺
69 591
1141
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
4767
⫺
14 927
2467
⫺
⫺
2684
⫺
⫺
⫺
⫺
⫺
6128
2808
10 417
⫺
31 185
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
III-20
III-25
III-37
III-38
III-39
PSA-Eu
⫺
⫺
466 040
⫺
1157
⫺
⫺
⫺
⫺
⫺
⫺
⫺
1855
⫺
⫺
⫺
144 901
15 737
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
2978
⫺
⫺
⫺
⫺
17 555
⫺
⫺
⫺
101 174
⫺
⫺
1463
⫺
⫺
⫺
6172
⫺
⫺
⫺
⫺
⫺
⫺
205 955
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
108 687
⫺
⫺
⫺
⫺
⫺
⫺
289 969
213 806
110 126
256 147
147 172
213 319
157 672
215 199
186 125
26 488
36 888
192 330
71 934
166 244
The assay was as described in footnote a of Table 2.
and hK2 (11, 12 ), further supporting the involvement of
these residues in the epitopes. Results from other groups
have suggested that most of the MAbs that bind both PSA
and hK2 recognize nonconformational linear epitopes
(35 ). The N-terminal part of PSA appears to form a fairly
long linear N-terminal epitope (residues 3–10) and thus
may be the major mediator of binding of MAbs in this
group. However, regions 3–10 and 138 –142 reside within
15–20 Å from each other, and amino acids within this
distance can be in contact with the paratope (37 ). Thus, it
is conceivable that the epitopes of some group VI MAbs
comprise residues 3–10 and 138 –142. MAbs 5E4 and H117
bind peptides similar to PSA 3–10 and 138 –142, but the
epitopes around amino acids 138 –142 are overlapping
rather than identical. Parhami-Seren et al. (42 ) also identified overlapping epitopes for two related MAbs against
streptokinase by screening peptide libraries.
Interestingly, in previous synthetic peptide epitope
mapping studies, MAb H117 did not react with any
synthetic linear peptide derived from the PSA sequence
(20 ), although it has been shown to compete efficiently
with other group VI MAbs (16 ). Thus, it is likely that the
synthetic peptides used by Piironen et al. (20 ) could not
adopt the correct conformation required for strong binding with MAb H117.
We also found some paratope-specific peptides showing sequence similarity with PSA, but the suggested
epitope locations were not in agreement with those in
previous studies. In a single peptide (III-8), the sequence
identity was not compatible with previous epitope assignments, suggesting that the sequence similarity was attributable to chance.
A key factor in the protocol for isolating peptides
resembling the linear epitope in the NH2 terminus of PSA
was the introduction of sequential panning with two
MAbs. With this method, peptides reacting with both
selection MAbs (5E4 and H117) were obtained. However,
with MAb pairs 9C5/H50 and 5A10/4G10, no peptides
reactive with both MAbs were found, which suggests that
the epitopes for these MAbs do not contain common
residues although they block the binding of each other.
Synthetic peptide mapping studies have suggested that
the binding sites for MAbs specific for free PSA reside in
the regions 50 – 69 (19 ) and 80 –91 (20 ). In the study of
Piironen et al. (20 ), all seven free-PSA MAbs reacted
strongly with peptides containing the 84 –91 sequence.
Some binding of MAbs was observed to peptides containing the sequence 50 – 67, but strong reaction with these
peptides was observed for only two MAbs recognizing
both free and complexed PSA. Furthermore, Jette et al.
(43 ) identified the sequence PSA 51–55 as an epitope for a
“total PSA specific” MAb by use of both synthetic peptide
epitope mapping and peptide library screening. Michel et
al. (21 ) suggested on the basis of peptide library screening
that the epitopes of MAbs specific for free PSA include
amino acid residues from PSA regions 53–59, 145–148,
and 204 –207. Our results also suggested that amino acids
204 –208 may form part of the epitope for such MAbs, but
no peptides resembling the two other sequences were
found. In the three-dimensional model, these amino acids
form a relatively wide loop, and it is possible that the
cyclic peptides used in our study are too tightly coiled to
mimic such a loop.
No peptides that bound strongly to MAb H50 were
found by Piironen et al. (20 ) by use of synthetic peptide
epitope mapping, but the binding sites of related MAbs
were mapped to the 151–155 region. We did not find any
peptides with similarity to this region by screening with
the two related MAbs, H50 and 9C5. However, a peptide
showing similarity with the 192–196 region was identified
after screening with MAb H50, and this region is adjacent
to region 151–155 in the three-dimensional model of PSA.
Furthermore, the 192–196 region is identical in PSA and
2215
Clinical Chemistry 48, No. 12, 2002
hK2, and MAb H50 is reactive with both PSA and hK2
(16 ).
reactivity of the peptides with isobm MAbs
Previous studies on reactivity of paratope-binding peptides with MAbs showing highly similar specificity suggested that peptides generally show restricted specificity
toward the selection MAb (44 ). This was also the case
with peptides to group I MAbs, which were specific for
free PSA, i.e., each peptide bound only to the MAb used
to isolate it. The peptides selected with group III MAbs
also showed restricted specificity. Most of them bound to
1–3 of the 14 MAbs specific for this region. In contrast, the
peptides selected with group VI MAbs recognized many
antibodies. Two peptides bound to one-half (7 of 13) of the
MAbs included. These results suggest that amino acids
3–10 form an immunodominant region that can be mimicked by the fairly short cyclic peptides. The restricted
specificity of the peptides to MAbs in other groups may
indicate larger variability in epitope specificity. Thus,
although some epitope regions can be subgrouped by use
of paratope-binding peptides, the general utility of this
approach for epitope identification is limited. Many peptides bearing no relationship with the immunogen (PSA)
bound strongly to the paratopes. Apparently these binding specificities are not displayed by natural human
antigens, as evidenced by the very high specificity of
MAbs when used for clinical purposes. However, the
unexpected reactivity of a MAb to BRCA1 with semenogelin may be an example of such polyspecificity (38 ). A
less dramatic but more common demonstration of this
polyspecificity is the nonspecific inhibition of antigenantibody binding exerted by proteins. This phenomenon
contributes to the well-known matrix effect that affects all
immunoassays (45 ). The nonspecific interactions between
various proteins in clinical samples and the paratope
must be much weaker that that of the specifically selected
peptides, and it can usually be eliminated by diluting the
sample (45 ).
In conclusion, this study shows that screening with peptide libraries can be used to define both linear and
noncontinuous epitopes, but the assignment of an epitope
region needs to be confirmed by complementary methods. Furthermore, although MAbs are highly specific
when used in clinical assays, the paratope region can bind
strongly to several unrelated peptide structures. However, strong nonspecific binding may be revealed only by
phage display peptides, whereas it appears to be very rare
in natural samples.
This work was supported by grants from the Academy of
Finland, Sigfrid Jusélius Foundation, and the Finnish
Cancer Society. We thank Liisa Airas and Maarit Leinimaa for expert technical assistance.
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