Epitope Mapping Protocols

406 Pages·1996·27.426 MB·English

by  Glenn E. Morris

Overview
Choosing a Method for Epitope Mapping
Glenn E. Morris
For most practical purposes, an epitope is easy to define as that part of an
antigen involved in its recognition by an antibody (or, in the case of T-cell
epitopes, by a T-cell receptor). Although simple chemical molecules, nucleic
acids, and carbohydrates can all act as antigens, the term “epltope mapping” is
usually applied to protein antigens, and is the process of locating the epitope
on the protein surface or m the protein sequence. The simplicity is deceptive,
however, and conceptual problems soon make their practical consequences felt.
A considerable understanding of the principles of protein structure and protein
folding, and some knowledge of the nature of the immune response may
quickly become necessary for the correct interpretation of experimental epitope
mapping results. The term “epitope mapping” has also been used to describe
the attempt to determine all the major sites on a protein surface that can elicit
an antibody response, at the end of which one might claim to have produced an
“epitope map” of the protein antigen (1). This information might be very use-
ful, for example, to someone wishing to produce antiviral vaccines. Implicit
in this view of epitopes is that they are fixed and concrete structures on
protein surfaces, which are few in number and uniquely capable of stimu-
lating the immune system. Even if this is true for proteins in their native con-
formation, it is a limitation imposed by protein structure rather than the immune
system, since additional immunogenic determinants are readily revealed by
protein unfolding. This kind of “epitope map” also confuses the important dis-
tinction between antigenicity (the ability to recognize a specific antibody) and
immunogenicity (the ability to produce antibodies in a given animal species).
Most people would agree that epitopes should be defined by their antigenicity.
From. Methods m Molecular Biology, vol 66, Epltope Mapping Protocols
Edited by* G E Morris Humana Press Inc , Totowa, NJ
2 Morris
It is essential to distinguish between conformational (“discontmuous,”
“assembled”) epitopes, m which amino acids far apart in the protein sequence
are brought together by protem foldmg, and lmear (“continuous,” “sequen-
tial”) epitopes, which can often be mimicked by simple peptide sequences.
Parts of conformational epitopes can sometimes be mimicked by peptides, and
the term “mimotope” has been coined to describe these peptides. On the other
hand, the view that most peptide sequences can produce antibodies that recog-
nize native proteins (2) has been disputed (‘3). Given the nature of protein struc-
tures, most epitopes on native proteins are likely to be “assembled” (4) and,
consequently, most antibody molecules m polyclonal antisera raised against
native protems do not recognize short peptides (‘3). If assembled epitopes are
found most frequently on native proteins, sequential epitopes are found more
often on denatured or partially unfolded proteins. Unfolding 1ss eldom, if ever,
complete under condttions conducive to antibody binding, since conditions that
unfold antigens (extremes of pH, chaotropic agents, ionic detergents, and so
forth) also affect immunoglobulins and antibody-antigen interactions. There is
something of a culture gap between crystallographers, who tend to study assem-
bled epitopes exclusively, and people who use monoclonal antibodies (MAbs)
as research tools, for whom assembled epitopes can be something of a nui-
sance if the MAbs do not work on Western blots. Some authors have preferred
to emphasize the distinction between epitopes on native protems and those on
denatured proteins by using such terms as “cryptotopes” or “unfoldons” for
the latter (5). Apart from their content, the titles alone of reviews by Laver
et al. (5) and Greenspan (6) are sufficient to illustrate the extent of this prob-
lem of definitions.
It might be simpler for the purposes of thus practical manual to adopt the
operational view that an epitope is defined by an antibody molecule, I.e., if an
antibody exists, then whatever it can be shown to recognize m the antigen is
the epitope (or part of it). This view has Its own problems, notably the fact that
MAbs often crossreact with sequences or structures other than that of the real
antigen. If it sidesteps many important issues (or brushes them under the car-
pet), it does at least recognize the fact that the extent of conformation depen-
dence of antibody binding is not always known when mapping begins. It also
implies that the number of epitopes could be as great as the number of antibod-
ies, depending on how often MAbs recognize identical epitopes. After stimula-
tion by antigen, a B-lymphocyte clone will undergo somatic mutation m a
germinal center of the spleen to refine antibody diversity further (7). The
slightly different antibody molecules produced in this way ~111g enerally rec-
ognize the same region of protein, but with a different affinity or a different
tolerance of amino acid substrtutions. These fine specificities can hardly be
regarded as defining different epitopes, although it is difficult to decide where
Overview 3
exactly to draw the line. At what point should the distinction between two
overlappmg epitopes cease to exist? Some may find such questions challeng-
ing, whereas others may find them merely tedious.
MAbs that bind to proteinso n Westernb lots (after SDS-PAGE) will tend to
be against sequential epitopes, whereas MAbs that recognize antigens in liq-
uid-phase immunoassays or in frozen tissue sections are often directed against
assembled epitopes. It must be remembered, however, that few proteins are
completely denatured on Western blots, and epitopes identified by Western
blotting may have a considerable conformational element. Another point
often overlooked is that the reducing agent (mercaptoethanol or dithiothreitol)
in SDS-PAGE may, for proteins with disulfide bridges, have a greater effect on
protein denaturation than SDS itself; for example, the binding of a number of
MAbs against hepatitis B surface antigen was retained after SDS treatment, but
abolished by reduction of the disulfide bridges that maintain the structure of
this antigen (81. MAbs can therefore be usefully divided into those that recog-
nize native proteins and are suitable for immunoassays, those that recognize
partially unfolded proteins and are suitable for Western blotting, and those that
recognize both. The antibody that defines an epitope will, of course, be an
MAb, the product of a single B-lymphocyte clone, although epitope mapping
methods can also be applied to polyclonal antisera, which should be regarded
as a mixture of MAbs. Consequently, unlike MAbs, antisera will usually rec-
ognize both native and denatured proteins, but different component antibodies
may be involved in the two cases; thus, the antibodies in an antiserum that are
used to demonstrate its specificity by Western blotting may be different from
those that are active in an immunoassay with that antiserum.
X-ray crystallography is often regarded as virtually the only method for pre-
cise definition of an epitope by identification of all the amino acids in contact
with the antibody. As Saul and Alzari show in Chapter 2, the contribution of
this technique to our understanding of epitopes has been outstanding. Its prime
position, however, is not completely unassailable for a number of reasons. First,
there does not seem to be complete agreement on how close amino acids in the
antibody and antigen must be to constitute a “contact.” Second, some residues
in the antigen could theoretically be “in contact” with the antibody without
contributing significantly to the binding. van Regemnortel has made the dis-
tinction between “structural” epitopes as defined by X-ray crystallography and
related techniques and “functional” epitopes defined by amino acid residues
that are important for binding and cannot be replaced (3). Third, the method is
restricted by the necessity of obtaining good crystals of antibody-antigen com-
plexes, and it has usually been applied to highly conformational epitopes on
the surface of soluble proteins. NMR methods in solution (Chapter 3) avoid the
need for crystals, but are limited by the size of the antigen that can be studied
4 Morris
and are usually applied to peptide antigens. Finally, the time and expense
involved in X-ray analysis tend to exclude it as a routine, everyday approach to
antibody characterization and epitope mappmg. Electron microscopy has also
been used successfully for low-resolution epitope mapping, although usually
for very large antigens, such as viruses (9). This method rather speaks for itself,
so a protocol has not been included.
Competition methods can be very useful when a relatively low degree of
mapping resolution is adequate. You may want to establish, for example, that
two MAbs recognize different, nonoverlapping epitopes for a two-site immu-
noassay, or to find MAbs against several different epitopes on the same anti-
gen so that results owing to crossreactions with other proteins can be rigorously
excluded. The principle behind competition methods is to determine whether
two different MAbs can bind to a monovalent antigen at the same time (in
which case they must recognize different epitopes) or whether they compete
with each other for antigen binding. Molinaro and Eby describe the simplest
possible method based on this principle, using Ouchterlony gel-diffusion
plates (Chapter 4); single MAbs or mixtures of MAbs that recognize overlap-
ping epitopes are unable to form precipitin lines. At a more sophisticated and
more expensive level, biosensors that follow antibody binding m real time
can be used to determine directly whether two or more unlabeled MAbs will
bind to the same unlabeled antigen. Johne describes the use of the Pharmacia
BIAcore for this purpose (Chapter 7). Such methods as ELISA using
microtiter plates are the traditional approaches to competition mapping, and
involve labeling either antibody or antigen with enzymeso r radioactivity. Kuroki
(Chapter 5) and Tzartos (Chapter 6) demonstrate the flexibility of this very
popular approach.
Chemical modification of amino acid side-chains is a method that is per-
haps less widely used today than previously (1). In principle, addition of
modifying groups specifically to amino acids, such as lysine, should prevent
antibody binding to epitopes that contain lysine residues, and such an
approach should be particularly useful for conformational epitopes that are
otherwise difficult to map with simple techniques (Chapter 8). Unfortunately,
such epitopes are also the most sensitive to indirect disruption by chemicals
that cause even small conformational changes, and great care is needed to
avoid false positives. The protection-from-modification method described by
Bosshard (Chapter 9) is more reliable in principle, since the side-chains in
the epitope itself are not altered (protected by antibody) and the modifying
groups on the unprotected side-chains are not large (e.g., radioactive acetyl
groups). Labeling of individual amino acids is compared in the presence and
absence of the protecting antibody. Protection from proteolytic digestion,
described by Jemmerson (Chapter lo), is similar in principle; for native pro-
Overview 5
teins, which are often resistant to proteases, it does depend on the epitope
containing a protease-sensitive site, but these, like assembled epitopes, are
often associated with surface loops. If the antibodyantigen interaction will
survive extensive proteolysis with loss of structure, the antigen fragments
remaining attached to the antibody can be identified by mass spectrometry
(Chapter 13). An alternative, and simpler, approach for epitopes that survive
denaturation is partial protease digestion of the antigen alone, followed either
by Western blotting for larger fragments or by HPLC (Chapter 12). The frag-
ments that bind antibodies can be identified by N-terminal microsequencing or
by mass spectrometry. Overlapping fragments, produced by different proteases,
help to narrow down the epitope location, Chemical fragmentation is an alter-
native to proteolysis and has the advantage that cleavage sites are less frequent
(e.g., for Cys, Trp, and Met residues) so that fragments can often be identified
from their size alone; for this reason, antigen purity is less important than for
proteolytic fragmentation (Chapter 11). Conditions for chemical cleavage,
however, are usually strongly denaturing, so the method is not useful for assem-
bled epitopes.
Synthetic peptides have revolutionized our understanding of epitopes to the
same extent as X-ray crystallography, although ironically the two approaches are
virtually mutually exclusive, since peptides are used for sequential epitopes.
Rodda et al. describe the PEPSCAN method in which overlapping peptides
(e.g., hexamers) covering the complete antigen sequence are synthesized on
pins for repeated screening with different MAbs (Chapter 14). Since the syn-
thesis can be done automatically, this popular approach requires very little work
by the end user (and, it sometimes seems, very little thought). The related
SPOTS technique for multiple peptide synthesis on a solid phase is described
by its originator in Chapter 15. An alternative approach to the synthesis of
peptides based on the antigen sequence is the use of libraries of completely
random peptide sequences. Pinilla et al. describe a method for the synthesis
and screening of such a library using their “positional scanning” approach in
Chapter 16. The advent of peptide libraries displayed on the surface of phage
(Chapters 17 and 18) took this approach a step further by enabling selection of
displayed peptides, as opposed to screening. In this case, random oligonucle-
otides are cloned into an appropriate part of a phage surface protein, and the
peptide sequence displayed is identified after selection by sequencing the phage
DNA. Selection of random peptides is unique in producing a range of sequences
that are related, but not identical, to the antigen sequence; this enables infer-
ences to be made about which amino acids in the epitope are most important
for antibody binding. An advantage shared by all peptide methods is that anti-
gen is not required, which may be important for “rare” antigens, which are
difficult to purify.
6 Morris
New possibilities for mapping arise if the antigen can be expressed from
recombinant cDNA. These include the mutation of ammo acids in the epitope,
and new methods of generating and identifying antigen fragments. Alexander
describes a method for altering individual amino acids in a known epitope by
oligonucleotide replacement (Chapter 20), and Shibata and Ikeda deal with the
introduction of random mutations into part of the antigen by PCR, followed by
screening to detect epitope-negative mutants (Chapter 2 1). An elegant method
for conformational epitopes, homolog scanning, described by Wang (Chapter
19) requires two forms of the antigen (e.g., from different species) to be
expressible from recombinant DNA as native proteins, one of them reactive
with the antibody and the other not. Functional chimeric proteins can then
be constructed by genetic engineering, and regions responsible for antibody
binding identified. Compared with random mutation methods, this approach is
less likely to disrupt the native conformation.
Two rapid methods for random shortening of the antigens produced from
plasmid vectors are described in Chapters 28 and 29. One of them takes advan-
tage of the spontaneous early termination of translation of mRNA, which
occurs in in vitro systems, whereas the other involves the random msertlon of
stop codons into plasmid DNA using a bacterial transposon. Extensive DNA
manipulation is not required m either method, although transposon mutagen-
esis has the additional advantage that the site of mtroduction of the stop codon
can be identified precisely by DNA sequencing. Brummendorf et al. describe
an elegant method for generating shortened fragments at both ends using exo-
nuclease III (Chapter 27). This enables production of overlapping fragments,
which can be used to determine epitope boundaries more reliably. As a bonus,
this chapter describes a novel vector, pDELF, specifically designed for map-
ping membrane proteins in mammalian cells. Random digestion of cDNA with
DNaseI, followed by cloning and expression, is a popular way of generatmg
overlapping antigenic fragments. Stanley described a method using bacterial
pEX plasmids in an earlier volume (10), so protocols for yeast plasmids (Chap-
ter 22) and bacteriophage h (Chapter 23) are described here. An additional
method for phage display of DNaseI fragments (Chapter 24) has the important
advantage that antibody-positive clones can be obtained by selection rather
than screening. In all cases, the antigen fragment expressed can be identified
by DNA sequencing.
Another approach is to clone specific, predetermined (rather than random)
fragments that have been generated either by using existing restriction enzyme
sites in the cDNA or, more flexibly, by using PCR products that have restric-
tion sites in the primers (Chapter 20). This approach is especially useful if you
want to know whether an epitope is in a specific domain of the antigen or
whether it is encoded by a specific exon in the gene, since other methods may
Overview 7
give ambiguous answers to these questions. For PCR products, the necessity to
clone may be avoided altogether by including a promoter in the forward primer
and transcrlbmg/translatmg the PCR product in vitro (Chapter 21). Another
major advantage of the PCR approach is that it is not always necessary to have
your full-length antigen already cloned. Provided the cDNA sequence is
known, reverse transcriptase-PCR (RT-PCR) can be used to clone PCR prod-
ucts directly from mRNA or even total RNA (II).
In Chapter 30, Rodda describes a synthetic peptide method for identification
of T-cell epitopes, and Chapter 3 1 is a simple reminder of the value of natu-
rally occurring sequence variants of antigens, such as isoforms or antigens from
different animal species, for identification of individual amino acid residues,
which may be important for antibody binding. Finally, the last two chapters
describe methods for generating the panels of MAbs that are needed for effi-
cient application of epitope mapping techniques. The traditional hybridoma
method has the advantage of 20 years of experience and refinement (Chapter
32), whereas phage display antibodies hold out the promise of better control of
antibody specificity and improved possibilities for “humanized” antibodies
(Chapter 33).
The choice of a method for epitope mapping depends on a number of fac-
tors, including:
1. The antigen: Is it available at all? In milligram quantities? As a recombinant
protein produced from cDNA7
2. The antibody: Does it recognizea n assembledo r a sequentiale pitopev
3. How detailed you want the mapping to be: Some methods identify individual
ammo acids essential for antibody binding whereas others only show whether
two epitopesa re sufficiently far apart for simultaneousb inding of the two anti-
bodies, with various levels of detail m between.
4. How much money/equipment/time you have available: Some of the methods
require expensive equipment that may not be readily available and other meth-
ods are heavy on both consumablesa nd time. Clearly, the most cost-effective
method will vary among laboratories.
If possible, it is advisable to give some thought to mapping problems at the
early stage of antibody production. I would recommend producing a panel of
different MAbs instead ofjust one or two, because some MAbs will invariably
prove easier to map than others. For detailed mapping at the amino-acid level,
it is easier to make antibodies that work on Western blots and probably recog-
nize sequential determinants. However, such antibodies may not be suitable
for all purposes, particularly immunoassay, for which they may lack the
reqmred specificity and high avidity. In my laboratory, we try to make MAbs
that recognize both native and denatured protein, but for many antigens, par-
ticularly globular proteins, this may prove extremely difficult or impossible.
8 Morris
Finally, where the methods in this volume require materials, such as plasmids
and bacterial strains, that are not available commercially, the chapter authors
will, in most cases, be happy to provide them.
Recent developments in epitope mapping include a method for displaying
peptide libraries directly on the surface of E. coli in the major flagellum com-
ponent, flagellin (12). Screening and amplification steps may be simpler than
in phage display and kits are available commercially (“FliTrx,” InVitrogen,
San Diego, CA). A commercial kit for expressing DNaseI fragment libraries in
bacteria is also available (“Novatope,” Novagen, Madison, WI). Another recent
development displays random peptide libraries on polyribosomes, and the selected
mRNA containing the peptide-encoding sequence is amplified by reverse tran-
scriptase-PCR for reselection or sequencing (13). Peptides have also been
chemically synthesized in very large numbers on microarrays for detection of
antibody binding by fluorescein-labeled second antibody and immunofluores-
cence microscopy (14).
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Overview 9
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