Synthetic Peptides for Antimucin Antibodies 129
129
From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
12
Synthetic Peptides for the Analysis and Preparation
of Antimucin Antibodies
Andrea Murray, Deirdre A. O’Sullivan, and Michael R. Price
1. Introduction
Since the mid-1980s, the family of high molecular weight glycoproteins known as
mucins have evoked considerable interest among those in the field of cancer research.
Mucins, which are constituents of mucus, have a lubricating and protective function in
normal epithelial tissue (1). However, expression of mucin by the cancer cell is often
highly disorganized and upregulated, sometimes to the extent that mucin can be
detected in the circulation of the cancer patient. These changes in expression of mucin
observed in neoplasia have led to the exploitation of some members of the mucin
family as circulating tumor markers (2,3) or targets for diagnostic imaging (4–6) and
therapy of cancer.
The first mucin to have its primary amino acid sequence determined, MUC1, is also
the most extensively studied. This molecule is highly immunogenic, and a consider-
able number of anti-MUC1 monoclonal antibodies (mAbs) and fragments have been
produced by various methods. Some of these have found applications for radio-
immunoscintigraphy and targeted therapy of cancer, and others have been used to
detect circulating MUC1. Although such studies have yielded promising results, their
present application is somewhat restricted. In this age of genetic and protein engineer-
ing, we have, at our disposal, the technology to design antibodies with ideal character-
istics of size, affinity, and specificity for any desired application. However, before
considering such ambitions, we must first gain an understanding of the molecular
interactions between epitope and paratope when an antibody binds to its antigen. It is
essential that key residues involved in the interaction are identified so that a model of
how the interaction takes place on a three-dimensional level can be constructed. This
identification will enhance our ability to design antibodies with the correct character-
istics for our chosen application.
130 Murray et al.
1.1. Immunoassays
Both enzyme-linked immunosorbant assays (ELISAs) and radioimmunoassays have
been used in various formats to test antibody binding to synthetic peptides. The indi-
rect ELISA has the advantages of being easy to perform, having no requirement for
radioactive tracers, and producing results that are simple to interpret. The disadvan-
tage of the indirect ELISA is that the procedure requires that the antigen, in this case a
synthetic peptide, be immobilized on to the surface of a microtiter plate well. Classi-
cally this would be achieved by dispensing a solution of antigen into the wells of a
microtiter plate to allow adsorption, leaving the plate coated with antigen. However,
short synthetic peptides adsorbed on to plates in this way provide unpredictable and
inconsistent results. This problem may be owing to the fact that the orientation of the
peptide on the plate cannot be controlled or simply that short peptides do not adhere
well to polystyrene plates. Several methods of peptide modification have been utilized
to overcome these problems. One such procedure involves preparing branched-chain
polypeptides in which MUC1 immunodominant peptides ware conjugated to a polyl-
ysine backbone (7). These polylysine conjugates provide very potent MUC1-related
antigens for the interrogation of antibody specificity; however, the methodology for
their preparation is beyond the scope of this chapter. By far the most widely used
method for modifying short peptides so that they can be used as antigens in indirect
ELISA procedures is to conjugate the peptides to a large carrier protein such as bovine
serum albumin (BSA) (see Subheading 3.1. and Notes 1–3).
1.2. Tethered Peptide Libraries for Exploring Antibody Specificity
The peptide synthesis techniques developed by Geysen and colleagues (8) repre-
sent a significant development in the study of epitopes defined by antibodies reactive
with antigens of known primary structure. Unlike most other methods of simultaneous
peptide synthesis, this technique allows the concurrent synthesis of hundreds to thou-
sands of peptides so that libraries can be produced and simultaneously used as targets
for antibody binding. The peptides are synthesized on derivatized polyethylene or
polypropylene gears that are held on stems (Fig. 1) arranged in a microtitre plate for-
mat so that a simple ELISA procedure can be used to measure antibody binding. Pep-
tides are tethered via the carboxyl terminus.
Several different strategies have been described for peptide sequence design that
all provide different information on epitope structure and the fine specificity of an
antibody-peptide interaction. The Pepscan approach has been the most widely used
and involves the synthesis of a set of overlapping peptides that span the length of the
antigenic sequence (Subheading 3.2.). In a short peptide sequence, such as that of the
MUC1 variable number of tandem repeat (VNTR), each peptide may overlap the next
by all but one amino acid, giving rise to a set of 21 heptapeptides that spans the VNTR
sequence (Fig. 2). For larger proteins, it is more appropriate to produce longer
sequences that overlap each other by less residues, thereby spanning the length of the
antigenic sequence with a feasible number of peptides (see Note 4). In the Pepscan
approach, peptides are assayed for antibody-binding capacity by ELISA (Subheading
3.3.), and residues that are common to all the antibody-binding pins represent the mini-
Synthetic Peptides for Antimucin Antibodies 131
Fig. 1. The Multipin Peptide Synthesis System contains detachable polyethylene gears that
fit on to the end of stems. The stems are held in a block in an 8 x 12 microtiter plate format. The
surface of the gear is derivatized to give a solvent-compatible polymer matrix on which the
peptides are coupled during synthesis. The matrix also provides a two amino acid spacer group.
Fig. 2. Schematic representation of the overlapping peptides corresponding to the MUC1
VNTR sequence synthesized according to the Pepscan approach to epitope mapping. Antibod-
ies are allowed to react with each peptide, and those containing the epitope or minimum bind-
ing unit produce positive results. In this example, the epitope can be deduced as consisting of
the amino acids that are common to all positive pins (7–10). Hence, the epitope is PDTR.
132 Murray et al.
mum binding unit or epitope for that antibody (Fig. 2). Having identified the epitope
defined by an antibody using Pepscan, it may be useful to prepare a number of analogs
of that sequence in order to investigate the role of individual amino acids in the epitope
and to identify critical contact residues. Such peptide design stategies include
ommission analysis, alanine substitution and replacement net (RNET) analysis (see
Notes 5–9).
Libraries of peptides on pins can be obtained that comprise 400 different dipeptides
prepared with all possible combinations of the 20 natural amino acids. This approach
provides qualitative information on antibody specificity and permits identification of
significant features of an epitope that may contribute to antibody recognition and bind-
ing (see Notes 10 and 11).
1.3. Purification of Antibodies Using Peptide Affinity Chromatography
The identification of a linear peptide epitope within a protein sequence facilitates
the design of peptide affinity matrices that can be used to purify antibodies from bio-
logical feedstocks. Such an epitope affinity matrix has been produced by covalently
linking a synthetic peptide corresponding to the MUC1-immunodominant domain to
cyanogen bromide-activated Sepharose (Pharmacia, Uppsala, Sweden) (9). The
resulting matrix was remarkably efficient for the purification of a range of anti-MUC1
mAbs from biological feedstocks containing high levels of contaminating proteins
such as ascitic fluid and hybridoma supernatant (see Note 12).
Epitope affinity chromatography matrices have an advantage over other affinity
adsorbents in that the antibody is bound to the matrix specifically via the paratope.
Thus, eluted antibody is fully immunoreactive and of only the desired specificity.
Sepharose-peptide conjugates are simple to prepare and affinity chromatography is
more robust than other conventional chromatographic techniques in terms of column
packing and operation (see Subheading 3.4., Notes 13–15, and Fig. 3).
1.4. General Comments
The techniques described for the analysis of antimucin antibodies using synthetic
peptides can provide a great deal of information on epitope topography and structure.
The identification of critical binding residues within an epitope can provide clues to
the forces and residues involved in the antibody-antigen interaction. However, bear in
mind that the use of linear synthetic peptides can only provide a one-dimensional
solution to what is essentially a three-dimensional problem. Further structural studies
such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and compu-
tational molecular modeling are essential if the knowledge gained is to be confirmed
and translated into a useful model on which to base antibody design strategies.
The structural information provided by studies such as those previously described
may be of use in peptide vaccine design. However, the analyses performed so far have
been mainly concerned with the interaction of murine antibodies, and it may be naive
to assume that the human immune system will process mucin-related antigens in the
same way. Preliminary epitope-mapping studies on human serum would suggest that
the immune response to MUC1 may differ considerably from that observed in the
mouse (10).
Synthetic Peptides for Antimucin Antibodies 133
Finally, it may be owing to the very nature of the mucins that such a wealth of informa-
tion has been provided by the techniques described. The VNTR provides a convenient
short sequence on which to base peptide synthesis strategies. In addition, most murine
antimucin antibodies analyzed to date have been shown to define short linear determi-
nants. It is unlikely that all other proteins and antibodies will be so accommodating.
Fig. 3. Schematic representation of the apparatus and reagents needed for the purification of
antibodies by peptide epitope affinity chromatography.
134 Murray et al.
2. Materials
2.1. Preparation of BSA-Peptide Conjugates
1. Conjugation buffer: sodium hydrogen carbonate buffer (0.1 M, pH 8.4).
2. BSA: crystalline, greater than 96% pure.
3. Glutaraldehyde: when used as a crosslinker must be freshly distilled or high commercial
grade (Sigma, Poole, UK).
4. Dialysis buffer: sodium chloride 1% (w/v).
2.2. Solid-Phase Peptide Synthesis on Pins
All reagents used in solid-phase peptide synthesis should be of the highest avail-
able purity (analytical reagent grade or better) unless stated otherwise.
1. Mulitpin Peptide Synthesis Kit (Chiron Mimotopes, Clayton, Victoria, Australia).
2. Amino acids: All amino acids recommended for use with the Multipin Peptide Synthesis
Kit have their α-amino group protected with the 9-fluorenylmethyloxycarbonyl (Fmoc)
group. Table 1 appropriate side chain protecting groups. Alternatively, protected amino
acid esters may be used. These have the advantage of requiring no prior activation. How-
ever, they are prone to decomposition with prolonged storage and are best stored at –20°C.
3. Activators: The activation of protected amino acids with diisopropylcarbodiimide (DIC)/
1-hydroxybenzotriazole (HOBt) is recommended, but other coupling reagents can be used.
4. N,N-Dimethylformamide (DMF): DMF used in peptide coupling procedures must be pure
and free from amines. Several methods may be used to purify DMF (see Note 16).
5. Piperidine 20% v/v: used for Fmoc deprotection. Piperidine should be redistilled before
use and made up to a 20% (v/v) solution in DMF.
6. Bromophenol blue: used as an indicator of coupling efficiency. Stock reagent is prepared
by dissolving 33.5 mg of bromophenol blue in 5 mL of DMF. This should be diluted
1:200 for working concentration.
7. Acetylation mixture: DMF, acetic anhydride and triethylamine in a 50:5:1 (v/v/v) ratio.
8. Side chain deprotection mixture: trifluoroacetic acid, ethanedithiol, and anisol in a 38:1:1 (v/v/v) ratio.
9. Final wash solution: acetic acid 0.5% (v/v) in methanol/water (1:1, v/v).
10. Other reagents: methanol (MeOH), purified water.
2.3. ELISA Testing Procedure
1. Phosphate buffered saline (PBS), 0.01 M, pH 7.2 (1.34 g of Na
2
HPO
4
·2H
2
O, 0.39 g of
NaH
2
PO
4
·2H
2
O, and 8.5 g of NaCl made up to 1 L with distilled water) is used as the
buffer base for most of the following buffer reagents.
Table 1
Suitable Amino Acid Side Chain Protecting Groups
for Solid Phase Peptide Synthesis on Pins
Side chain protecting group Amino acid
t-Butyl ether S, T, Y
t-Butyl ester D, E
t-Butoxycarbonyl K, H, W
2,2,5,7,8-Pentamethylchroman-6-sulfonyl R
Trityl C
Synthetic Peptides for Antimucin Antibodies 135
2. Blocking buffer: 2% (w/v) BSA, 0.1% (v/v) Tween-20, and 0.1% sodium azide in 0.01 M PBS.
3. Conjugate diluent: 1% (v/v) sheep serum, 0.1% (v/v) Tween-20, and 0.1% sodium casein-
ate (USB, Bioscience, Cambridge, UK) in 0.01 M PBS.
4. Citrate phosphate buffer: 17.8 g of Na
2
HPO
4
·2H
2
O and 16.8 g of citric acid monohydrate
made up to 1 L with distilled water, pH 4.0.
5. 2,2'-azino-bis[3-ethylbenz-thiazoline-6-sulfonic acid] (ABTS) substrate solution (Sigma):
0.5 mg/mL in citrate phosphate buffer with hydrogen peroxide (35% w/w) added to give
a final concentration of 0.01% (w/v).
6. Disruption buffer: Sodium dihydrogen orthophosphate (0.1 M) pH 7.2, containing sodium
dodecyl sulfate (SDS) (0.1% w/v). β-Mercaptoethanol (5 mL) is added immediately prior to use.
2.4. Purification of Antibodies Using Peptide Affinity Chromatography
1. Affinity support: CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden).
2. Equilibration buffer: 0.01 M PBS with azide (PBSA), pH 7.2 (1.34 g of Na
2
HPO
4
·2H
2
O,
0.39 g of NaH
2
PO
4
·2H
2
O, and 8.5 g of NaCl made up to 1 L with distilled water) with
sodium azide 0.02% (w/v) added as a preservative.
3. Wash buffer: 0.5 M NaCl, pH 7.2, in distilled water.
4. Elution buffer: 3 M NaSCN, pH 7.2, in distilled water.
5. Desalting column: Sephadex G25 (Pharmacia).
3. Methods
3.1. Preparation of BSA-Peptide Conjugates (
see
Notes 1–3)
1. Dissolve BSA (10 mg) in 3 mL conjugation buffer in a clean glass vial.
2. Dissolve peptide (10 mg) in 1 mL conjugation buffer.
3. To the BSA solution, add 1 mL of peptide solution and 10 µL of glutaraldehyde. Then
seal and agitate on a roller for 4 h at room temperature.
4. The conjugate is finally dialysed against sodium chloride (1%) for 48 h at 4°C.
3.2. Solid-Phase Peptide Synthesis on Pins (
see
Notes 4–11, and 17)
The Multipin Peptide Synthesis Kit (Chiron Mimotopes) contains derivatized gears,
stems, 8 × 12 format pin holders, and reaction trays. In addition, it contains all the
software needed for creating a synthesis schedule, running dispensing aids, and read-
ing and plotting assay results.
3.2.1. Creating a Synthesis Schedule
The method described by Geysen et al. (8) for linear epitope scanning requires that
many different peptides be synthesized simultaneously. To plan and execute a manual
synthesis schedule for creating hundreds of peptides simultaneously is extremely time-
consuming and fraught with the possibility of errors. Fortunately, computer software is
available to generate a synthesis schedule based on any given protein sequence with any of
the manipulations described (Chiron Mimotopes). These schedules calculate the weights
and volumes of the various reagents required on each day of the synthesis and then instruct
the operator where in the 96-well reaction tray each amino acid should go (see Notes 5–8).
3.2.2. Peptide Synthesis
The peptide synthesis procedure consists of cycles of N-terminal deprotection,
washing and coupling until the desired peptides have been assembled, followed by
136 Murray et al.
side chain deprotection. The synthesis schedule provides details of the amounts of
amino acids and activators required. It is advisable to weigh out all these reagents
before beginning a synthesis since this is the most time-consuming step of the proce-
dure. All steps are carried out at room temperature unless stated otherwise.
1. The appropriate number of gears required for that synthesis on that day should be removed
from storage and assembled on to the block according to the synthesis schedule. It is impor-
tant that only gears requiring deprotection in the next cycle of synthesis be added to the block.
2. Achieve deprotection of the amino terminus by immersing the pins in a bath containing
20% piperidine for 20 min. The piperidine solution should cover the gears. The pins are
then washed as follows:
a. DMF to cover gears for 2 min.
b. MeOH (complete submersion) for 2 min.
c. MeOH to cover gears for 2 min (three times).
The pins are then allowed to air-dry in an acid-free fume hood for a minimum of 30 min.
3. Prepare HOBt and DIC solutions by dissolving in the appropriate amount of DMF (see
Note 13). The addition of bromophenol blue to the HOBt to give a final concentration of
0.05 mM as an indicator of coupling efficiency is optional. The volume of HOBt solution
specified on the synthesis schedule must be added to each amino acid to dissolve it fully
before adding the specified amount of DIC.
4. Dispense amino acid solutions into a 96-well reaction tray according to the synthesis
schedule. The recommended order of activating and dispensing amino acids is as follows:
A D E F G I L M P S T V Y W Q N K C H R. Care should be taken to ensure that the
amino acids are dispensed into the correct wells. Dispensing aids are now available that
consist of a bank of LED lights set out in a microtiter plate format. Lights are lit beneath
the reaction tray to indicate which wells should contain which amino aid. The dispensing
aid is driven by the synthesis schedule software.
5. Place the block of Fmoc-deprotected pins into the reaction tray in the correct orientation.
Place the tray into a polystyrene box to reduce evaporative losses and avoid contamina-
tion and leave to incubate for at least 4 h.
6. When the coupling reaction is complete, the blue colouration of bromophenol blue should
have disappeared. The pins are then washed as follows:
a. MeOH to half the pin height for 5 min.
b. air-dry for 2 min.
c. DMF to half the pin height for 5 min.
The next cycle of peptide synthesis can begin immediately with Fmoc deprotection.
7. When the required peptides have been synthesized, deprotect and wash the free amino
termini as described in step 2. The amino terminus may then be acetylated to remove the
charge associated with a free amino terminus (if required) by incubating the pins in a
reaction tray containing acetylation mixture at 150 µL/well for 90 min in an enclosed
environment. Then wash the pins in MeOH for 15 min and then air-dry.
8. To achieve side chain deprotection, incubate the pins in a bath of side chain deprotection
mixture for 2.5 h. Next, immerse in a final wash solution for 1 h, rinse twice in MeOH for
2 min each, and air-dry overnight. The pins are now ready for ELISA testing.
3.3. ELISA Testing Procedure (
see
Notes 7 and 12)
Antibody binding to peptides on pins is measured using an indirect ELISA proce-
dure in which the solid phase on which the test antibody is captured is the peptide-
Synthetic Peptides for Antimucin Antibodies 137
coated gear, and the presence of the test antibody is reported using an enzyme-labeled
secondary antibody. The enzyme catalyzes the reaction of ABTS substrate to its col-
ored product, which can be measured using a spectrophotometer. The degree of color
change is proportional to the amount of test antibody bound to the peptide on the gear.
Before antibody testing begins, the newly synthesized pins should be tested for non-
specific binding to the enzyme-labeled secondary antibody of choice. This is achieved
by carrying out steps 1 and 4–6. Antibodies may now be tested as follows, with all
incubations and washing steps performed at room temperature unless otherwise stated.
1. First precoat the pins in blocking buffer in order to minimize nonspecific binding to the
gear. To achieve this, immerse the pins in a microtiter plate containing blocking buffer at
200 µL/well and incubate for 1 h with agitation.
2. Dilute the primary antibody to an appropriate concentration in blocking buffer and dispense
into the wells of a microtiter plate at 200 µL/well. After removing from blocking buffer and
flicking to remove excess buffer, incubate the pins in primary antibody at 4°C overnight.
3. Remove the pins from the microtiter plate and wash four times in a bath of PBS contain-
ing Tween-20 (0.1% v/v) for 10 min. Use fresh buffer for each wash.
4. Dilute an appropriate horseradish peroxidase-labeled secondary antibody conjugate (e.g., horse-
radish peroxidase-conjugated rabbit antimouse Ig is suitable for detecting murine primary anti-
bodies) in conjugate diluent and dispense into the wells of a microtiter plate at 200 µL/well. Then
incubate the washed pins in the secondary antibody solution for 1 h with agitation.
5. Wash the pins four times as in step 3. Prepare ABTS substrate solution immediately
before use and dispense into the wells of a microtiter plate at 200 µL/well. Immerse the
pins in the substrate solution in the correct orientation and allow to incubate for 45 min
with agitation. The reaction can be stopped before the time is elapsed, if it appears that
the reaction will give an optical density (OD) of 2 or greater, by removing the pins from
the wells and then allowing the microtiter plate to shake for a further 15 min to allow full
color dispersion. The OD of each well is determined spectrophotometrically at a wave-
length of 405 nm.
6. Bound antibodies can be removed from the pins by sonication in disruption buffer at
60°C for 2 h, followed by repeated rinses in distilled water at 60°C and methanol (two
times). The efficiency of the cleaning procedure should be tested by repeating steps 4 and
5. Absorbance levels above background indicate that antibody remains bound to the pins
and further cleaning is required. Once the pins are clean, they should be sonicated for 30
min followed by rinsing as detailed just above (see Note 17).
3.4. Purification of Antibodies Using Peptide Affinity Chromatography
(
see
Notes 12–15 and Fig. 3)
1. Prepare Sepharose-peptide affinity matrices and pack columns according to the manufacturer’s
instructions (see Notes 12 and 13).
2. Equilibrate the columns with 10 column volumes of PBSA at a flow rate of 1 mL/min.
3. Clarify hybridoma or bacterial culture supernatants by ultracentrifugation (40,000g, 1 h)
and ultrafiltration (0.2 µm) and then store with 0.05% (w/v) sodium azide as a preservative.
4. Apply clarified supernatant to the column at a rate of 1 mL/min followed by washing with
PBSA, to remove unbound material, until the trace from the ultraviolet monitor has re-
turned to baseline.
5. Optional: Wash the column with 0.5 M NaCl (1 mL/min) to remove material that has
bound to the column nonspecifically.
138 Murray et al.
6. To achieve desorption of specifically bound, pure antibody, apply three column volumes
of 3 M NaSCN to the column at a rate of 1 mL/min and finally return the column to PBSA
(see Notes 14 and 15).
7. Antibody preparations desorbed using 3 M NaSCN must be desalted soon after elution
from the affinity matrix. To achieve this, connect a gel filtration column containing a
medium such as Sephadex G25 in series with the affinity column (Fig. 3).
4. Notes
1. Conjugation of peptides to carrier proteins can also be performed in situ in the well of a
microtitier plate (11).
2. Synthetic peptide-carrier protein conjugates and synthetic branched-chain polypeptides
provide highly characterized and reproducible sources of mucin-like antigenic material.
However, bear in mind that these reagents are analogs of the natural antigen and do not
possess the carbohydrate side chains that are a dominant characteristic of all mucins.
Hence, results obtained in immunoassays, especially those involving the measurement of
kinetic data, must be treated with caution.
3. The influence of carbohydrates on the recognition of peptide epitopes may be evaluated,
at least to some extent, using synthetic glycopeptides rather than peptide alone. Glyco-
peptides can be produced by both chemical (12) and enzymatic (13,14) methods. These
reagents have been of value in assessing the contribution of O-linked N-acetyl-
galactosamine (GalNAc) residues to mucin secondary structure and also in the studies to
investigate the role of GalNAc residues in the binding of protein core antibodies (15).
However, the glycosylation of mucin molecules is complex, and the production of higher-
order synthetic mucin analogs with more than a single sugar at each glycosylation site is
technically demanding.
4. The length of peptides synthesized seems to have no effect on the result obtained as dem-
onstrated by two independent studies of anti-MUC1 protein core mAbs. One group used
heptamers spanning the tandem repeat domain and overlapping each other by six amino
acids (16), and the other used octamers overlapping each other by seven amino acids (17).
Twelve antibodies were analyzed in total and the three that were analyzed in both studies
gave identical minimum binding units.
5. In the omission analysis approach, a series of peptides are synthesized based on an epitope
sequence. In each consecutive peptide, a single residue is omitted from the sequence.
This allows the role of individual residues to be assessed. For example, an omission analy-
sis series covering the immunodominant region of the MUC1 protein core may be synthe-
sized. If an antibody were allowed to interact with this series of peptides, those
sequences that produced a loss in binding compared with the parent sequence can be
identified as part of a peptide in which an essential residue has been omitted. Antibody
binding is maintained in those peptides in which the epitope is complete.
6. In the substitution analysis approach, each residue is replaced in turn with another amino
acid. This amino acid is normally alanine, but for cases in which an alanine already exists
at that substitution position, the residue can be replaced with glycine. The information
provided when an antibody is allowed to react with this set of peptides is similar to that of
omission analysis; however, in this case, the spatial arrangement of the respective epitope
residues more closely resembles the native sequence.
7. RNET analysis offers the most critical and informative method for probing a peptide
epitope in the MUC1 protein core. In this approach, a set of peptides with sequences
based on a short minimum binding sequence or epitope are synthesized on the heads of
Synthetic Peptides for Antimucin Antibodies 139
pins. Each residue within the epitope is systematically replaced by each of the other natu-
rally occurring amino acids to provide a library of peptides with mutations at specific
points. For example, for a tetrapeptide epitope consisting of the amino acids RPAP, the
RNET would consist of four sets of 20 peptides with the general sequences: XPAP,
RXAP, RPXP, and RPAX, where X is any naturally occurring amino acid.
8. Using RNET analysis, it is possible to identify residues that cannot be replaced by any
other and thus are essential for antibody binding. Such residues almost certainly have a
role in forming the correct three-dimensional structure of the epitope and may also pro-
vide the forces between epitope and paratope required to maintain binding. Conversely, it
is possible to identify residues that are present in the epitope but can be replaced by other
amino acids of similar size and charge. These can be assumed to play no direct role in
antibody binding but may serve to restrict the conformation of the epitope so that the
critical contact residues are presented in the optimal orientation for antibody binding.
9. RNET analysis has also provided information on the role of residues flanking the
epitope that are not involved in antibody binding but are required for epitope presen-
tation. Briggs et al. (18) studied the HMFG-2 antibody, which defines the minimum
binding unit DTR, using a set of RNET peptides based on a PDTR tetrapeptide. These
studies revealed that the antibody required the presence of the proline residue at posi-
tion 1 for binding even though this residue was not found to be part of the minimum
binding unit using Pepscan.
10. Care should be taken when interpreting the results of assays using dipeptides since ELISA
signals from the best binding pairs may be low compared with those obtained for “com-
plete” epitopes (19) In addition, the very nature of the library facilitates the binding of
any antibody, including the second antibody conjugate used as a reporter molecule in the
ELISA procedure. Careful control experiments must therefore be performed in order to
allow differentiation of signals produced by binding of the test antibody and false-posi-
tive signals caused by the reporter antibody binding directly to the pins.
11. A dipeptide library may also be used to probe the specificity of antibodies against pro-
teins whose primary structure is unknown as well as to identify peptide mimotopes for
antibodies whose antigen is not protein in nature.
12. A column comprising of MUC1 immunodominant peptide conjugated to Sepharose 4B
was able to adsorb in excess of 40 mg of IgG/mL of affinity matrix, resulting in a concen-
tration factor of about 1000. Antibodies prepared in this way were found to be pure enough
for administration to humans (9). A matrix such as this one has since been used for the
purification of recombinant anti-MUC1 Fv fragments (20,21) as well as human anti-
MUC1 antibodies from serum (Murray, A., unpublished findings).
13. A detailed study of the fine specificity of the reaction of an antibody with its antigen can
lead to the design of peptide ligands with improved purification performance. For in-
stance, it has been found that the C595 mAb has higher affinity for a synthetic MUC1
glycopeptide compared with the naked peptide. This information has been used to design
a matrix consisting of a diglycosylated MUC1 glycopeptide linked to Sepharose, which
demonstrates improved antibody yields in affinity chromatography experiments (12).
14. A variety of reagents can be used to achieve desorption of immunoreactive antibody.
Consult standard chromatography text for further details.
15. NaSCN gradient elution provides a simple method for determining the relative affinities
of a series of antibodies for a matrix or a single antibody for different matrices (12). The
technique relies on the observation that a higher-affinity interaction would require a
greater concentration of chaotrope to effect desorption of antibody. The use of an auto-
140 Murray et al.
mated fast protein liquid chromatography system allows the production of accurate gradi-
ents that can be measured with a conductivity monitor. Manual gradient elution requires
more effort and patience but can be achieved successfully with practice.
16. Distillation in vacuo results in high-purity DMF, which can be stored for up to 2 wk if
kept under nitrogen in dark bottles at 4°C. Alternatively, stand over an activated molecu-
lar sieve (4 Å) for several days and then filter off the DMF. Use within 2 d.
17. A major advantage of the technique is that the peptides are covalently bound to the gears,
so that harsh conditions such as sonication in SDS and mercaptoethanol can be used to
remove bound antibodies after testing without affecting the peptide. Hence the peptide
libraries can be reused to test other antibodies. In our experience, these immobilized pep-
tide arrays have been used to test 50 or more antibodies with no loss in activity.
Reference
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