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ORIGINAL ARTICLE
Positive reactions on Western blots do not necessarily
indicate the epitopes on antigens are continuous
Yi-Hua Zhou, Zhaochun Chen, Robert H Purcell and Suzanne U Emerson
Epitope mapping (identification of an antigenic site recognized by an antibody) is an important component of vaccine
development and immunological assays. It is widely accepted that in Western blots, antibodies react exclusively with continuous
epitopes: discontinuous epitopes are assumed to be irreversibly destroyed by electrophoresis under the denaturing conditions
used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Here, we demonstrate that the epitopes recognized by four
different monoclonal antibodies were identified as discontinuous epitopes when characterized by radioimmunoprecipitation
assays and enzyme-linked immunosorbent assays, yet each of these antibodies reacted with the corresponding antigen on
Western blots. Reaction on Western blots may be due to epitope renaturation during or after the transfer of the protein to a
membrane. Therefore, positive reactions on Western blots do not necessarily indicate that epitopes are continuous and this
caveat should be kept in mind while characterizing them.
Immunology and Cell Biology (2007) 85, 73–78. doi:10.1038/sj.icb.7100004; published online 28 November 2006
Keywords: SDS-PAGE; immunoblotting; epitope type
Antibodies are used for various purposes, not only by immunologists
but also by biologists from a wide range of disciplines. Identification
of immunoreactive subregions on antigens recognized by antibodies,
referred to as epitope mapping, is a critical step that is helpful in
developing immunoassays, generating vaccines, studying protein–
protein interactions, defining protein topology and investigating the
pathogenesis of autoimmune diseases, etc. Epitopes are divided into
two categories: continuous (linear, sequential) epitopes, which are
formed by a contiguous segment of the amino-acid sequence, and
discontinuous (conformational, assembled) epitopes, in which amino-
acid residues far apart in the primary sequence are brought together
to assemble a topographic site on the surface of a protein.1
In epitope mapping, the first step is usually to distinguish between
continuous and discontinuous epitopes by Western blots. It is widely
accepted that discontinuous epitopes are denatured during prepara-
tion for sodium dodecyl sulfate (SDS)-polyacrylamide gel electro-
phoresis (PAGE), thus rendering them unrecognizable by antibodies.
Therefore, antibodies that bind to proteins on Western blots are
considered to recognize continuous epitopes. This hypothesis, how-
ever, has resulted in numerous contradictory or incomplete data in the
literature about the nature of specific epitopes.
Many authors have used a series of overlapping synthetic peptides
covering the partial or complete antigen to define the continuous
epitopes that their antibodies reacted with on Western blots. In many
cases, however, the antibodies did not bind to any synthetic peptide.
While some authors suggested the epitopes might therefore be
discontinuous,2,3 others still concluded that the epitopes were con-
tinuous.4–9 Although the failure to map the epitopes was explained by
the splitting or cleavage of the epitope during the preparation of
synthetic peptides,4,6 such speculations were not confirmed. Therefore,
these data appear to contradict the concept that antibodies reactive on
Western blots are always directed to continuous epitopes.
Many researchers have also used truncated polypeptides produced
in various expression systems to map epitopes. Some of them claimed
to have localized the epitopes according to the reaction pattern of the
antibody with polypeptides truncated at only one terminus, either at
the N or C terminus, thus neglecting the possible role of distant amino
acids in forming the epitope(s).10–16 Apparently, they assumed that the
antibodies reactive on Western blots recognized continuous epitopes
and that the lack of reactivity of antibodies with polypeptides
sequentially truncated from one terminus was sufficient to determine
the location of the epitopes. However, these data may be incomplete.
It was reported that Western blots may be used to exclusively detect
antibodies to continuous epitopes on B19 parvovirus VP1 and VP2
structural proteins;17,18 however, these epitopes have not been defined.
There are multiple other examples of epitopes determined by Western
blots to be continuous without any confirmative evidence that
was so.19–28
In characterizing monoclonal antibodies (mAbs) that reacted with
hepatitis E virus (HEV) capsid protein on Western blots, we tried to
Received 16 June 2006; accepted 20 July 2006; published online 28 November 2006
Laboratory of Infectious Diseases, Hepatitis Viruses and Molecular Hepatitis Sections, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
MD, USA
Correspondence: Dr Y-H Zhou, Laboratory of Infectious Diseases, Hepatitis Viruses and Molecular Hepatitis Sections, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Building 50, Room 6535, 50 South Drive MSC-8009, Bethesda, MD 20892, USA.
E-mail: yzhou@niaid.nih.gov
Immunology and Cell Biology (2007) 85, 73– 78
&
2007 Australasian Society for Immunology Inc. All rights reserved 0818-9641/07 $30.00
www.nature.com/icb
determine the epitopes using random peptide libraries; however,
no reactive peptide was identified. This led us to question whether
Western blot-reactive antibodies exclusively recognize continuous
epitopes. Here we present evidence that some discontinuous epitopes
may be detected by Western blotting analysis. The widely held notion
that Western blot-reactive antibodies are directed to continuous
epitopes is indeed a serious misconception, resulting in futile studies
using sophisticated and expensive technologies to define continuous
epitopes that do not exist.
RESULTS
Polypeptides recognized by mAbs EBL5 and EBL89
In a previous study, EBL5 and EBL89 were found to compete with
each other in enzyme-linked immunosorbent assay (ELISA) and to
react with the N-terminal region of HEV capsid protein.29 To further
define the region recognized by each mAb, we prepared a series of
35S-labeled second open reading frame (ORF2) polypeptides truncated
at the N or C terminus (Figure 1a) and tested them by radioimmuno-
precipitation assay (RIPA) for reactivity with the mAbs. Some poly-
peptides appeared as doublets on SDS-PAGE, which might reflect
premature termination. Figure 1b shows that, in the native RIPA
buffer, EBL5 precipitated most of the ORF2 polypeptides, including
those spanning amino acids 112–320 and 141–446, but did not
precipitate the polypeptides spanning amino acids 112–280, 112–260
or 151–446. Therefore, the polypeptide region recognized by EBL5 had
an N terminus close to amino acid 141 and a C terminus close to
amino acid 320, and contained approximately 180 amino acids,
suggesting that the epitope was discontinuous. Similarly, mAb
EBL89 precipitated six of the 10 ORF2 polypeptides in the native
RIPA buffer, including those covering amino acids 112–320 and
131–446, but did not precipitate polypeptides with further terminal
truncations (Figure 1b). Therefore, the region recognized by EBL89
(amino acids 131–320) was very similar in size to the region
recognized by EBL5, again suggesting that the recognized epitope
was discontinuous.
To further characterize the reactivity of EBL5 and EBL89, we mixed
them with the ORF2 polypeptides in the denaturing RIPA buffer.
As shown in Figure 1b, EBL5 reacted with only three polypeptides
mAb EBL5
HEV ORF2
aa1 aa660
446
408
360
320
280
260
121
131
141
151
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
64
50
36
22
16
kDa
64
50
36
22
16
kDa
mAb EBL89
Native
112
112
112
112
112
112
446
446
446
446
64
50
36
22
16
kDa
12345678910
Denaturing
12345678910
Native
12345678910
Denaturing
12345678910
12345678910
a
b
Figure 1 Polypeptides recognized by mAbs EBL5 and EBL89 in radioimmunoprecipitation assays. (a) Diagram of HEV ORF2 protein and
35S-labeled polypeptides. The numbers beside each bar indicate the first and last amino acid (a.a.) of each polypeptide. The polypeptides were labeled with
35S-methionine during in vitro translation, separated by electrophoresis, and detected by autoradiography. Lanes 1–10 correspond to polypeptides numbered
1–10 in parentheses. (b) Precipitation of 35S-labeled polypeptides by mAbs EBL5 and EBL89. Each polypeptide was incubated with each mAb in native and
mildly denaturing buffers (see Methods), respectively. The immune complexes were collected on protein G-coupled agarose. The precipitated polypeptides
were identified by SDS-PAGE and autoradiography. Lanes 1–10 on each gel correspond to polypeptides 1–10 in (a).
Discontinuous epitopes on Western blots
Y-H Zho u et al
74
Immunology and Cell Biology
(112–446, 121–446 and 131–446) and the reaction was weak; EBL89
did not react with any polypeptide under denaturing conditions.
These patterns of reactivity were considerably different from those
in the native RIPA buffer (Figure 1b).
Polypeptides recognized by mAbs EBL16 and EBL56
A previous study showed that both EBL16 and EBL56 reacted with the
C-terminal region of HEV capsid protein, but they did not compete
with each other,29 indicating they recognized non-overlapping epi-
topes. To further localize the epitopes, we prepared a second series
of 35S-labeled ORF2 polypeptides truncated at the N or C terminus
(Figure 2a) and tested them by RIPA for reactivity with the mAbs. As
shown in Figure 2b, both mAbs reacted with all polypeptides contain-
ing amino acids 459–607 of ORF2 protein, but further truncation
by removal of nine residues from the C terminus (112–598) or five
residues from the N terminus (464–607) abolished the reactivity.
Therefore, the polypeptide recognized by EBL16 and EBL56 contained
between 149 and 135 amino acids (459–607 and 464–598, respec-
tively), suggesting that the epitopes recognized by EBL16 and EBL56
were discontinuous. Interestingly, the patterns of reactivity of both
mAbs in the mildly denaturing RIPA buffer were the same as those
in the native RIPA buffer (Figure 2b).
Reactivity of EBL5, EBL89, EBL16 and EBL56 on Western blots
Previously, we prepared in bacteria three truncated polypeptides,
containing amino acids 110–458, 458–607 and 475–607 of HEV
ORF2, respectively.33 In the present study, we generated and purified
an additional polypeptide containing amino acids 458–598 of ORF2.
The identity of each polypeptide was confirmed by Western blots
using a mouse mAb against a 6-mer histidine tag at the N terminus of
each polypeptide (Figure 3a). Combinations of the four bacterially
produced ORF2 polypeptides and an insect cell-produced polypeptide
covering amino acids 112–607 of ORF2,37 which had been used as the
panning antigen in the production of EBL5, EBL89, EBL16 and
EBL56, were subjected to SDS-PAGE under denaturing and reducing
conditions and then blotted to membranes. The ORF2 polypeptides
on the membranes were probed by each mAb, respectively (Figure 3b).
Both EBL5 and EBL89 reacted with two polypeptides (112–607 and
110–458), but did not react with a polypeptide covering amino acids
458–607 (Figure 3b). These results were consistent with the epitope
mapping results of RIPA, which demonstrated that the epitopes
recognized by EBL5 and EBL89 were within polypeptides, including
amino acids 141–320 and 131–320, respectively (Figure 1b). On the
other hand, EBL16 and EBL56 reacted with two polypeptides
(112–607 and 458–607), but did not react with two other shorter
HEV ORF2
aa1 aa660
459
598
464
449
429
479
(1)
(2)
(3)
(4)
(5)
(7)
(6)
64
50
36
22
16
kDa 123456 7
kDa
mAb EBL16
mAb EBL56
112
112
607
607
607
607
607
607
Native
64
50
36
22
16
kDa
64
50
36
22
16
1234567
Native Denaturing
1234567 1234567
Denaturing
1234 5 6 7
a
b
Figure 2 Analyses of epitopes recognized by mAbs EBL16 and EBL56 by radioimmunoprecipitation assays. (a) Diagram of HEV ORF2 protein and
35S-labeled polypeptides. See legend to Figure 1. (b) Precipitation of 35S-labeled polypeptides by mAbs EBL16 and EBL56. See legend to Figure 1.
Discontinuous epitopes on Western blots
Y-H Zho u et al
75
Immunology and Cell Biology
polypeptides (458–598 and 475–607) (Figure 3b). These results were
also in agreement with the epitope mapping results of RIPA, which
localized the epitope to amino acids 459–607 (Figure 2b). Addition-
ally, the demonstration that EBL16 and EBL56 did not react with
polypeptides 458–598 and 475–607 confirmed the specificity of
positive reactions on the Western blots.
Reactivity of EBL5, EBL89, EBL16 and EBL56 in ELISAs
To further characterize the epitopes recognized by these mAbs, we
compared the reactivity of each mAb diluted in phosphate-buffered
saline (PBS) or in mildly denaturing RIPA buffer in ELISAs based on
the N- and C-terminally truncated polypeptides. As shown in Figure 4,
each mAb diluted in PBS reacted with the coated ORF2 polypeptides
that had been identified as containing the corresponding epitope, but
did not react with any further truncated polypeptides. On the other
hand, while either EBL16 or EBL56 had the equivalent reactivity in
denaturing RIPA buffer, and in PBS, EBL5 showed significantly
reduced reactivity and EBL89 did not react with any polypeptide in
EBL89
EBL16 EBL56
EBL5
64
50
36
22
16
110 −
−
458
112 − 607
112 − 607
458 − 607
475 − 607
458 − 598
112 − 607
458 − 607
475 − 607
458 − 598
110 − 458
458 − 607
112 − 607
110 − 458
458 − 607
458 − 607
475 − 607
458 − 598
kDa
Anti-His6
64
50
36
22
16
kDa
64
50
36
22
16
kDa
a
b
Figure 3 Reactivity of mAbs analyzed by Western blots. All polypeptides
were boiled for 5min in Laemmli buffer. The numbers above each lane
indicate the first and last amino acid of each HEV ORF2 polypeptide.
(a) Western blots detected by anti-His6. The polypeptides, which were
synthesized in E. coli, have a 6-histidine tag at the N terminus. The bands
were visualized by adding 3,3¢-diaminobenzidene solution. (b) Western blots
with mAbs against HEV. Polypeptide 112–607 was produced in insect
cells.37 The bands were visualized by enhanced chemiluminescence.
OD450OD450
Coated peptide
Coated peptide
Coated
p
e
p
tide
0
0.5
1.0
1.5
2.0
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
EBL5 EBL89
0
0.5
1.0
1.5
2.0
2.5
3.0
OD450
0
0.5
1.0
1.5
2.0
2.5
3.0
EBL16 EBL56
Anti-His6
112 − 607
112 − 607
110 − 458
458 − 607
475 − 607
458 − 598
458 − 607
475 − 607
458 − 598
112 − 607
458 − 607
475 − 607
458 − 598
112 − 607
110 − 458
110 − 458
458 − 607
458 − 607
Figure 4 Reactivity of mAbs against HEV in ELISA under native and mildly
denaturing conditions. The first and last amino acid of each HEV ORF2
polypeptide is given. Polypeptides 110–458, 458–607, 458–598 and 475–
607 have a 6-histidine tag at the N terminus. Open bar represents mAbs
that were diluted in PBS. Hatched bar represents mAbs that were diluted in
denaturing buffer (see Methods).
Discontinuous epitopes on Western blots
Y-H Zho u et al
76
Immunology and Cell Biology
denaturing buffer, whereas they both reacted with two polypeptides in
PBS (Figure 4). To exclude the possibility that the reduced or lack of
reactivity was caused by detachment of the polypeptides coated on the
ELISA plate under the denaturing conditions, we tested the reactivity
of a mouse mAb against the histidine tag, which was located at the N
terminus of each polypeptide, in PBS and denaturing RIPA buffer,
respectively. As shown in Figure 4, the reactivity of the anti-His6to
each ORF2 polypeptide in PBS and in denaturing RIPA buffer was
equivalent, indicating the ORF2 polypeptides were still adsorbed to
the ELISA plate under the denaturing conditions.
DISCUSSION
In the present study, we found that four mAbs that recognized
discontinuous epitopes reacted with the corresponding proteins on
Western blots after the proteins were subjected to SDS-PAGE under
denaturing and reducing conditions. The epitopes recognized by our
four mAbs were deemed to be discontinuous based on the large
number of amino acids required for the polypeptides to be reactive. In
the RIPA under the native conditions, the smallest tested polypeptides
recognized by mAbs EBL5, EBL89, EBL16 and EBL56 contained
approximately 180, 190, 149 and 149 amino-acid residues (Figures
1b and 2b). These results indicated that the epitopes recognized by the
mAbs were unlikely to be continuous because the binding area on the
hypervariable domains of an antibody is fully occupied by at most 15–
22 amino-acid residues.38 Additionally, under even mildly denaturing
conditions, mAbs EBL5 and EBL89 showed significantly decreased or
no reactivity to the corresponding proteins in RIPAs and ELISAs
(Figures 1b and 4). The complete agreement between the reactivity of
each mAb in RIPAs and in ELISAs provided compelling evidence that
the epitopes analyzed in the present study were discontinuous rather
than continuous.
The results of Western blot analyses in this study demonstrated that
discontinuous epitopes could be recognized by the mAbs after the
antigens had been separated by SDS-PAGE under denaturing condi-
tions. On Western blots (Figure 3b), all mAbs reacted with the
corresponding antigens used in the selection of these antibodies as
well as with the epitope-containing polypeptides that were reactive in
the RIPA or ELISA, but they did not react with shorter polypeptides.
The mechanism permitting the recognition on Western blots
following denaturation is not yet known. Some polypeptide antigens
may be somewhat resistant to denaturing conditions. mAbs EBL16
and EBL56, which recognized the epitopes formed by the C-terminal
region (amino acids 459–607) of ORF2, were equally reactive in the
presence of 1% SDS and under native conditions (Figures 2b and 4).
However, resistance to denaturation cannot fully explain the reactivity
on Western blots as, under denaturing conditions, mAbs EBL5 and
EBL89 displayed decreased or no reactivity in either RIPA or ELISA
(Figures 1b and 4), yet both reacted in Western blots (Figure 3b).
Thus, the recognition of discontinuous epitopes on Western blots
must reflect other mechanisms.
It is more likely that the denatured polypeptides in the gel might
renature enough to reform some epitopes during and/or after transfer
to the membranes.39 During the transfer, the protein-bound SDS
would have gradually decreased as the transfer buffer used did not
contain SDS. After the transfer, the proteins on the membranes were
no longer under denaturing conditions in any subsequent step.
Therefore, denaturation of protein antigens in SDS-PAGE may not
irreversibly destroy all discontinuous epitopes.
The renaturation of discontinuous epitopes on Western blots after
denaturing SDS-PAGE apparently is not a rare event. Previously, we
found that two Western blot-reactive mAbs against HEV also recog-
nized discontinuous epitopes.32,33 Additionally, we have found that
epitopes recognized by eight other mAbs against HEV (data not
shown), a mAb against vaccinia virus40 and a mAb against protective
antigen of anthrax (data not shown) were all discontinuous, although
these mAbs reacted with the corresponding antigens on Western blots.
Some epitopes reported in the literature to be continuous may actually
be discontinuous as they could not be defined by overlapping
synthetic peptides.
In conclusion, Western blots must be interpreted with caution
and an epitope should not be considered continuous until reaction
with a short peptide is confirmed.
METHODS
Antibodies
The four mAbs used in the present study (EBL5, EBL16, EBL56 and EBL89) are
against HEV capsid protein,29 which is encoded by the ORF2. All four mAbs
were generated by phage display libraries from bone marrow of chimpanzees
infected with HEV and subsequently immunized with the ORF2 protein
produced in a baculovirus expression system.
Radioimmunoprecipitation of 35S-labeled polypeptides by mAbs
An RIPA was used to localize the polypeptide recognized by each mAb. A series
of ORF2 fragments differing in size were amplified from HEV Sar-5530 cDNA
in the plasmid pHEV63.231 by polymerase chain reaction (PCR). The PCR
products were purified and inserted into T-tailed pGEM-T vectors (Promega,
Madison, WI, USA) by the T–A overhang cloning method. 35S-labeled ORF2
polypeptides were prepared with the TNT T7/SP6-coupled in vitro transcrip-
tion/translation system (Promega) using 35S-methionine.
RIPA was performed essentially as described previously.32,33 Briefly, a
mixture of a 35S-labeled polypeptide and an mAb was mixed with an equal
volume of 2native (0.5 MNaCl, 20% glycerol, 1% Tween 20 and 2 mM
ethylenediaminetetraacetic acid, 0.2 MTri s , p H 7 . 4 ) or 2 mildly denaturing
(0.3 MNaCl, 2% SDS, 2% Triton X-100, 2% deoxycholate, 0.1 MTris, pH 7.4)
RIPA buffer and incubated at 41C overnight. It should be noted that the sample
was not heated so the secondary structure of some proteins might be retained
even in the presence of SDS.34–36 The immune complexes were collected with
secondary anti-human immunoglobulin (Ig)G F(ab¢)2 (Pierce, Rockford, IL,
USA) and protein G-coupled agarose beads (Amersham, Piscataway, NJ, USA),
washed with RIPA buffer, eluted with denaturing Laemmli buffer by boiling for
5 min and subjected to 16% Tris/Glycine gel (Invitrogen, Carlsbad, CA, USA).
The 35S-labeled polypeptides were detected by autoradiography.
Preparation of an HEV ORF2 polypeptide in Escherichia coli
A truncated polypeptide spanning amino acids 458–598 on the HEV ORF2
protein was generated essentially as described elsewhere.33 The recombinant
polypeptide, which contained a histidine tag and an Xpress epitope at the N
terminus, was purified with the ProBond Purification System (Invitrogen)
using nickel-chelate affinity chromatography via the hexa-histidine tag. Its
identity was confirmed by Western blots using horseradish peroxidase (HRP)-
conjugated mAb against the hexa-histidine tag. The polypeptide was dialyzed
against PBS (pH 8.6–9.0) to allow renaturation and its concentration was
determined with the Micro BCA Protein Assay Reagent Kit (Pierce).
Western blots
Each of the polypeptides was mixed with an equal volume of 2Laemmli
buffer (4% SDS, 10% mercaptoethanol, 20% glycerol, 0.004% bromophenol
blue and 0.125MTris-HCl, pH 6.8), boiled for 5min and subjected to SDS-
PAGE (16% Tris-Glycine gel, Invitrogen). After electrophoresis in a denaturing
running buffer (0.1% SDS), the polypeptides were transferred to nitrocellulose
membranes by electroblotting in Tris/Glycine buffer (Invitrogen). The mem-
branes were blocked in 0.05% Tween 20 in PBS containing 5% skim milk
powder for 1h and incubated with anti-His6or the m Abs f or 1 h. After
extensive washes, the membranes were incubated for 30 min with a HRP-
conjugated anti-mouse or anti-human IgG F(ab¢)2 (Sigma, St Louis, MO, USA)
diluted 2000- or 10 000-fold in PBS. The protein bands were visualized by
Discontinuous epitopes on Western blots
Y-H Zho u et al
77
Immunology and Cell Biology
adding 3,3¢-diaminobenzidene (Sigma) or by enhanced chemiluminescence
(Pierce) followed by autography.
Enzyme-linked immunosorbent assay
An indirect ELISA was used to detect the reactivity of each mAb to correspond-
ing polypeptides essentially as described elsewhere.33 The reactivity was tested
under native (antibodies were diluted in PBS) and denaturing (antibodies were
diluted in mildly denaturing RIPA buffer containing 1% SDS) conditions,
respectively.
ACKNOWLEDGEMENTS
This work was supported by the Intramural Research Program of the National
Institutes of Health, National Institute of Allergy and Infectious Diseases.
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