1961
ClinicalChemisty 42:121961-1969 (1996) Enzyid1
Epitope mapping of prostate-specific antigen withmonoclonal antibodies
DIANE C. JETTE,’ FERNANDO T. KREUTZ,1 BRUCE A. MALCOLM,’- DAVID S. WISHART,2
ANTOINE A. NOuJAIM,3 and MAVANUR R. SURESHI*
Prostate-specific antigen (PSA) is a widely used marker forscreening and monitoring prostate cancer. We identified
and characterized the epitopes of two anti-PSA monoclonal
antibodies (niAbs) designated B80 and B87. The epitopeswere initially mapped as nonoverlapping by developing a
sandwich inimunoassay to measure PSA with the two anti-
PSA mAbs. The two antibodies do not cross-react withhomologous pancreatic kallikrein, but recognize epitopes
unique to PSA. B80 and B87 can recognize both free andcomplexed PSA and hence measure total PSA. Epitopescanning and bacteriophage peptide library affinity selec-tion procedures were used to identify and locate an epitopeon PSA. A possible epitope for B80 was identified as being
located on or near PSA amino acid residues 50-58 (-GRH-
SLFHP-). The epitope for B87 was likely on an exposednonlinear conformational determinant, unique to PSA, and
not masked by the binding of B80 or a1-antichymotrypsin.
INDEXING TERMS: immunoassay #{149}prostate cancer . bacterio-phage peptide library
Prostate-specific antigen (PSA) is a 33-kDa single-chain glyco-
protein produced by prostatic epithelium.4 The primary struc-ture of PSA was first described by Watt et al. [1] and shows a
high degree of homology with serine proteases of the kallikrein
family [2, 3]. PSA is a major protein in seminal plasma and shows
chymotrypsin-like substrate specificity [4-6/. It is normally
Faculty of Phannacy and Pharmaceutical Sciences, and 2 1)eparrment of
Biochemisny, Faculty of Medicine, University of Alberta, Edmonton, AB, Canada
T6G 2N8.Biomira Research Inc., Edmonton, AB, Canada.
Author for correspondence. Fax 403-492-8241; e-mail [email protected].
ualberta.ca.
Nonstandard abbreviations: PSA, prostate-specific antigen; BPII, benign
prostadc hyperphasia; inAb, inonoclonal andbody; hK2, human glandular kal-
hikrein; hKh, human pancreatic kallikrein; AMG, a2-macroglohulin; ACT, o,-
antichymotrypsin; I-IRPO, horseradish peroxidase; hsmAb, bispecific niAb; BTS
2,2-azino-bis(3-ethylbcnz-thiazoline-6-suhfonic acid); SDS, sodium dodecyl sul-
fate; PAGE, polyacrylamide gel electrophoresis; TMB, tetramethylbenzidine;
PBS, phosphate-buffered saline; BSA, bovine serum albumin; pKl, porcine
pancreatic kalhikrein; and TBS, Tris-buffered saline.
Received January 16, 1996; revised June 25, 1996; accepted June 25, 1996.
found in low concentrations (<2.5 jigfL) in male blood plasmabut can be increased in conjunction with prostate cancer, benignprostatic hyperplasia (BPH), and surgicaltrauma to the prostate
[7-9].Monoclonal antibody (niAb)-based RIAs and ELISAs are
used to measure PSA in serum. The results are used to screen for
prostate cancer and monitor patients during treatment[8, 10, 11].
The PSA gene is a member of the human tissue kallikrein
gene family referred to as serine proteases because the mecha-nism of proteolytic cleavage involves a serine residue at the
active site [1, 12, 13]. PSA is a 6-kb gene product of chromo-
some 19 in the region of q 13.2-q 13.4 with 4 introns and 5 exons[14]. It has a >84% nucleotide sequence homology with humanglandular kallikrein (hK2), and 73% homology with human
pancreatic kallikrein (hKl), indicating a common ancestral gene[3, 15-18]. Human serum kallikrein is not a member of this gene
family and does not have a high degree of sequence homology to
PSA. The amino acid homology with porcine pancreatic kal-
likrein(pKl) is59.6%. Previously all three gene products were
renamed [15, 17], as shown in Table 1.
In serum, PSA can form stable complexes with two majorserum protease inhibitors, a,-macroglobulin (AMG) and a1-
antichymotrypsin (ACT) [20-22]. When PSA iscomplexed to
AMG it is thought to be completely encapsulated with noepitopes accessible for immunodetection [23]. However, when
PSA is complexed to ACT, some epitopes proximal to the activesite are masked while others are available for binding to
anti-PSA antibodies [21]. Immunoassays involving antibodies
directed against PSA epitopes that are masked by ACT will failto detect this complex. The proportion of serum PSA corn-plexed to ACT has heen found to be significantly higher withprostate cancer than with BPH. The ratio of free to complexed
PSA, therefore, may be useful in distinguishing between pros-tatic cancer and BPH [21-23]. It is important, therefore, to
determine if antibodies used to measure PSA in serum arerecognizing free PSA, PSA-ACT, or both. In addition, several
authors have recently discussed the importance of standardizingPSA assays, because many of the commercially available assays
yield different PSA values [24-30]. In this paper, we attempt toidentify and characterize the epitopes that are recognized by two
anti-PSA mAbs.
1962 Jetteet al.:PSA epitopemapping
Table 1. Nomenclature of kallikrein family proteins.aFormal name Commonname Description
hKl PRK Human pancreatic/renal kallikrein,hKLK1 gene product
hK2 hGK-1 Human glandular kallikrein, hKLK2gene product
hK3 PSA PSA, hKLK3 gene product
pKl Porcine pancreatic kallikrein
Modified from Mccormack et al. [19].
Materials and MethodsMaterials. Anti-PSA mouse IgG mAbs B80 and B87 were kindly
provided by Biomira, Edmonton, AB, Canada, and the anti-PSA X anti-peroxidase (HRPO) bispecific mouse IgG mAb
(bsmAb B8OXHRPO) was developed in our laboratory [31].Briefly, a quadroma or hybrid hybridoma was generated by
fusing the B80-secreting hybridoma with an anti-HRPO-secret-ing hybridoma. The bsmAb was purified by DE-52 gradient
chromatography. This bsmAb has one Fab arm with anti-PSAspecificity and another Fab arm with anti-HRPO specificityengineered in one molecule. Standard media used for cellculture, RPMI-1o40 supplemented with 2 mmnol/L L-glutamine,50 kilounits/L penicillin, 50 mg/L streptomycin, and 50 mL/L
fetal bovine serum, were obtained from Gihco BRL, Gaithers-burg, MD. Anti-mouse and anti-rabbit IgG-peroxidase conju-
gate, cyanogen bromide-activated Sepharose 4B, pKl, kallikreinfrom human serum, and 2,2’-azino-bis(3-ethylbenz-thiazoline-
6-sulfonic acid) (ABTS) were obtained from Sigma, St. Louis,MO. Maxisorp” 96-well immunoplates were obtained fromNunc, Roskilde, Denmark. PSA calibrator was obtained from
Scripps Labs., San Diego, CA. ACT from human plasma andrabbit anti-ACT IgG were obtained from Calbiochem, La Jolla,CA. Molecular-mass markers for sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE), precast 4% to15% gradient polyacrylamide gels, SDS buffer cartridges, andthe Phast electrophoresis system were obtained from Pharma-
cia, Uppsala, Sweden. The Mimotope kit, used to synthesize theiinniobilized hexapeptides, was obtained from Cambridge Re-
search Biochemicals, Cheshire, UK. A Vmax microplate reader
and MAX Line plate washer from Molecular Devices, MenloPark, CA, was used. Escherichia coli strain K 91 (Hfi-C thi) was
used in conjunction with the bacteriophage (phage) decapeptide
display library [32J. Dideoxmucleotide sequencing was per-formed with the Sequenase 2.0 system obtained from UnitedStates Biochemicals, St. Louis, MO. One Step TMB (tetram-ethylbenzidine) substrate was obtained from Pierce Chemical
Co., Rockford IL.
Purification of PSA. A human prostate cancer cell line (LNcap)was used as a source of PSA. The cells were grown (standardmedia, 275 mm2 tissue culture flask) and the supernatant was
collected and pooled every 3 to 5 days. The concentration ofPSA in the unpurified supernatant was determined to be 0.9-1.5irmg/L by ELISA. PSA was purified from I L of cell supernatant
by using an affinity column with immobilized anti-PSA antibod-
ies, B80 and B87. The column was made by first washing
Sepharose 4B (1 g in 3.5 mL, cyanogen bromide activated) with2 volumes of buffer (0.5 mol/L phosphate, pH 6.8). A mixture of
B80 and B87 [10 mg in 3 mL of phosphate-buffered saline(PBS), pH 7.21 was dialyzed overnight against three changes of
buffer. The antibodies were added to the Sepharose gel and
gently agitated at room temperature. The amount of antibodyremaining in solution was determined by monitoring the reac-
tion mixture absorbance at 280 nm. The reaction was stoppedwhen -90% of the antibody was bound. The column was then
washed twice with PBS (10 mL), and ethanolamine (10 mL of100 ninol/L in distilled water) was added to block any unbound
reaction sites on the Sepharose (2 h at room temperature).Before use, the mAb affinity column was washed twice (10
mL of 10 mmollL phosphate buffer, pH 6.4). Cell supernatant
containing 1.5 mg/L PSA (1 L) was cycled for 24 h through thecolumn with a peristaltic pump (1.7 mL/min) so that the totalvolume of supernatant passed through the column three times.
After washing the column with buffer, the PSA was eluted with
glycine (100 mmol/L, pH 2.5). Column fractions (0.5 mL) were
collected into tubes containing buffer (50 .tL of 1 mol/L
phosphate, pH 8.0) to neutralize the eluates. The proteinconcentration of each fraction was determined by measuring theabsorbance at 280 nni and the appropriate fractions werepooled. After pooling, an ELISA was performed as describedbelow to determine the concentration of purified PSA. The
column, which can be reused, was washed (1 g/L sodium azide
in 10 mmol/L phosphate) and stored at 4 #{176}Cuntil needed. PSA
eluted from the monoclonal affinity column in fractions 3 to 5and a total of 1.37 mg of PSA was recovered from 1.5 L of
supernatant. No remaining PSA was detected in the supernatantafter affinity purification.
Preparation and analysis of PSA -ACT complex. The complex was
prepared by using the method described by Lilja et al. [22].Purified PSA (7 g) was added to purified ACT (50 g) in
reaction buffer (50 iL of 50 mmol/L Tris-HC1, pH 7.8, with
0.1 molJL NaC1) and incubated for 6 h at 37 #{176}C.The degree of
complex formation was analyzed by SDS-PAGE. A Phast elec-trophoresis system was used to analyze free and complexed PSA.
A precast aciylamide gel (4% to 15% gradient) was run under
standard conditions (10 mA, 250 V in SDS-barbital buffer) as
per the manufacturer’s instructions. Samples containing 4 [IL of
purified PSA, complexed PSA, unbound ACT, and low-rangemolecular-mass markers were analyzed. SDS-PAGE could ef-
fectively resolve PSA at -30 Wa, ACT at -60 Wa, andPSA-ACT complex at -90 Wa. A small amount of free PSAwas seen on the electrophoresis gel after complex formation.The PSA-ACT complex immunoassay used in this study will not
detect free PSA or ACT.
MEASUREMENT OF PSA AND PSA-ACT BY ELISA
PSA sandwich ELISA. We previously developed two mouse
mAbs, B80 and B87, using human PSA isolated from seminal
plasma. These two antibodies were selected for their ability toform an efficient sandwich assay for PSA in serum. The
antibodies have high affinity for PSA (Kd -2 X o o mol/L forboth) and the assay exhibited a good correlation (r = 0.98) with
50 100 150 200 250
Antibody concentration (pg/L)
Clinical Chemistry 42, No. 12, 1996 1963
the Hybritech PSA Tandem RIA with 154 prostate cancer
serum samples (Krantz MJ and Suresh MR, manuscript inpreparation). A PSA sandwich ELISA was developed with mAbB87, as the capture antibody, immobilized on plastic in a
microtiter plate. The bsmAb B8OXHRPO was used as the tracer
antibody [31].Microtiter wells were coated with mAb B87 (1 tg
in PBS, 16 h, 4 #{176}C).The plate was then blocked [10 g/L bovine
serum albumin in PBS (BSA-PBS), 1 h, 37 #{176}C}.Samples orcalibrators (50 [IL) were added to each well along with bsmAb
B8OXHRPO (10 ng) and HRPO (300 ng in 50 [IL of PBS). Theplate was incubated with shaking (30 mm, room temperature).
After washing [3 x 200 [IL of PBS containing 0.5 mL/L Tween20 (PBS-Tween)], ABTS substrate (100 [IL of 0.5 g/L ABTS,
0.5 [IL of 30% H,O,, in 0.1 mol/L citrate with 0.1 mollLphosphate, pH 4.0) was added to each well and the amount of
PSA present in the saIiple was determined by measuring theabsorbance at 405 nm with a microplate reader. The assay wascalibrated for PSA concentration with 99% pure PSA obtained
commercially. The calibrators were diluted in BSA-PBS (andnot serum). This assay was used to determine the PSA concen-
tration in cell supernatant and PSA affinity-purified eluates. Thedose-response curve, shown in Fig. 1, is linear from 0 to 75[Lg/L PSA and is not saturated even at 250 [Ig/L PSA. Each
point is an average of duplicate measurements and the SEM isindicated. Further optimization and the clinical utility of this
assay are currently being evaluated in our laboratory [31 and
work in progress]. Another experiment directly compared thequantitative recovery of fixed samples of PSA ranging from 3 to
100 [Ig/L with and without ACT in molar ratios of 1:0, 1:0.5,1:1, and 1:5. The absorbance values obtained at 100 [Lg/L were
1.286, 1.210, 1.296, and 1.206 and at 6 [Ig/L were 0.107, 0.086,
0.112, and 0.097 for the respective ratios. The absorbance values
similarly were within experimental error for 3, 12, 25, and 50
[Ig/L PSA with and without ACT in the above ratios.
2.5
2.0
1.5
10
0.5
0 50 100 150 200 250
PSA (pg/L)
300
Fig. 1. Dose-response curve for a PSA sandwich ELISA.mAb B87, immobilized on the plate, was used as the capture antibody andbsmAb B8OXHRPO was used as the tracer antibody. Pure PSA, diluted in 10 g/LBSA-PBS, was used to calibrate the assay for PSA concentration. The assay wasperformed as described in Materials and Methods.
PSA -ACT ELISA. Microtiter wells were coated with either B80
or B87 (1 g in PBS, overnight, 4 #{176}C)and blocked as above.
PSA-ACT complex was titrated from 100 ng/well (100 [IL
BSA-PBS) in twofold serial dilutions. The plate was incubated
(shaking, 1.5 h, room temperature) and then washed. Anti-ACT
rabbit IgG polyclonal antisera was diluted (100 [IL, 1:1000
dilution in BSA-PBS) and added to each well. The plate was
again washed. Anti-rabbit IgC-HRPO was diluted as above and
added to each well. The plate was incubated for I h and washed.
ABTS substrate was added and the amount of immobilized
PSA-ACT complex present in the sample was determined by
measuring the absorbance at 405 nm.
Cross-reactivity analysis. Human serum kallikrein and pKl were
immobilized on plastic microtiter wells. B80 or B87 was incu-
bated with the immobilized proteins to evaluate their cross-
reactivity. The binding of these antibodies to immobilized PSA,shown in Fig. 2, reaches saturation in this assay at -12 [Lg/L for
B80 and 50 [Ig/L for B87. No binding to either of the kallikreins
was observed for B80 or B87 up to a concentration of 1 mg/L.
Purified PSA was used as a positive control and BSA was used as
a negative control. Microtiter wells were coated with either
purified PSA, hK2, or pKl (0.5 g, PBS, overnight, 4 #{176}C).Theplates were blocked, and B80 or B87 was titrated from 1 [Ig/well
in BSA-PBS in twofold dilutions. The plate was incubated for
3 h, then washed. Anti-mouse IgG-HRP() was diluted as above
and added to each well. The plate was incubated for I h and
washed. ABTS substrate was added and the amount of antibody
binding to kallikreins or PSA was determined by measuring the
absorbance at 405 nm.
EC
0
0
Fig. 2. Comparison of the binding of mAbs B80 and 687 to PSA.glandular (or human serum) kallikrein, or pKl in an immunoassay.PSA or kallikrein was immobilized on the plate. After incubating with B80 or B87.the plate was washed to remove unbound material, and anti-mouse lgG-HRPOconjugate was used as the secondary antibody. Neither B80 nor B87 cross-reacted with either kallikrein. The assay was performed as described in Materialsand Methods.
1964 Jette et al.: PSA epitope mapping
SYNTHESIS AND SCREENING OF PSA HEXAPEPTIDES
The 240 overlapping hexapeptides representing the entire PSA
sequence were synthesized on pins with the Mimotope kit.
Standard solid-phase peptide synthesis with Fmoc-amino acid
active esters was used to build each hexapeptide on amino-functionalized pins. The peptides were acetylated at the N
terminus. The pins with the immobilized synthetic peptides can
be regenerated and retested several times.A standard ELISA was used to screen the PSA-derived
peptides for binding to B87 and bsmAb B8OXHRPO. The pins
were blocked in a 96-well microtiter plate. For screening with
B87, antibody (100 ng/well, 200 [IL of BSA-PBS) was added to
each well of a microtiter plate and the pins incubated in thissolution for 3 h and washed. Anti-mouse IgG-HRPO was
diluted and added to each well of a microtiter plate. The pinswere again incubated and washed. ABTS substrate was added
and the amount of antibody binding to immobilized peptide was
determined by measuring the absorbance at 405 nm. For
screening with B80, bsmAb B8OXHRPO (10 ng) and HRPO(300 ng in 200 [IL of PBS) were added to each well. The pins
were incubated for 1 h and subsequently washed. ABTS sub-strate was added and the absorbance at 405 nm was measured.
AFFINITY SELECTION OF B80 AND B87 SPECIFIC PEPTIDES
FROM A RANDOM PHAGE PEPTIDE DISPLAY LIBRARY
An aliquot of a bacteriophage peptide library displaying random
decapeptides [32] [4.4 X 10’’ colony-forming units in 40 [IL of
BSAJTris-buffered saline, pH 7.2 (BSA-TBS)I was added to 40
g of immobilized B80 and B87 (10 [IL of a 50% slurry of
antibody-linked Sepharose 4B, described above). The sample
was gently mixed (room temperature, 1.5 h) and the beads were
collected by ccntrifugation (30 s, 4000g). The supernatant was
removed and the beads resuspended, washed with 500 [IL ofBSA-TBS (5 nun), and recovered by centrifugation. This wash
was repeated two times with BSA-TBS, three times with 0.5
mL/L Tween 20 in TBS (TBS-Tween), and once with BSA-TBS. The washed beads were then incubated with PSA (1 h,
room temperature with shaking, 3 g in 50 [IL of BSA-TBS).
The supernatant, designated as ligand eluate, was collected and
the beads washed as before with 500 [IL of 10 mmollL citrate
buffer, pH 2.0. After 5 mm, the supernatant, designated as the
acid eluate, was collected. Both the ligand and acid eluates wereamplified by infecting E. co/i cells with isolated phage, following
procedures described in Christian et al. [32]. A second round ofselection was performed as above.
IMMUNOLOGICAL SCREENING OF AFFINITY-SELECTED PHAGE
Affinity-selected phage particles were screened in an in situ
immunological assay with phage-infected colonies [32]. Phage
clones from the eluates were used to infect fresh cells. The cellswere then grown in selective media (15 X 150 mm plates, LB
medium with 20 mg/L tetracycline) at a density of 200 to 500
colonies/plate and transferred to nitrocellulose membrane fil-
ters. For screening with B80, the filters were washed (30 mm,TBS-Tween), blocked (30 mm, 30 mL/L BSA-PBS), and thenincubated with bsmAb B8OXHRPO (20 g, with 1 mg of
HRPO). The filters were washed again and incubated with
TMB substrate for I h. The filters were then washed with
distilled water and allowed to air dry overnight. Alternatively,
filters with colonies from a second set of plates were incubated
with B87 (1 mg in 100 mL of BSA-PBS) for 2 h at room
temperature. After washing, the B87 filters were incubated with
anti-mouse-HRPO (1:1000 dilution in BSA-PBS, I h, room
temperature). The filters were washed three times and incubated
with TMB substrate. After 1 h the filters were washed with
distilled water and allowed to air dry overnight. Positive clones
were picked from the plates and amplified for sequencing.
Dideoxynucleotide sequencing was performed by using the
Sequenase 2.0 system according to the manufacturer’ s instruc-
tions.
MODELING OF PSA
A homology model of PSA was generated with INSIGHT II
molecular modeling package (Biosym Technologies, San Diego,
CA). The model was prepared by initially aligning the human
PSA sequence against those of both porcine kallikrein (59%
sequence identity) and rat tonin (54% sequence identity) by
using structural alignment routines in the SEQSEE software
[33] package (Biotools, Edmonton, AB, Canada). On the basis ofthese two alignments and on the quality of the available
structural data, the x-ray structure of rat tonin (Brookhaven
Protein Datahank accession code: ITON) was selected as the
three-dimensional template for homology modeling the PSA
structure. By using the sequence alignment produced by SEQ-
SEE, the amino acid sequence of PSA was then substituted into
that of tonin by using the Biopolymer module of INSIGHT II.
The model was subjected to a brief period of conjugate gradient
energy minimization to reduce unfavorable van der Waals
contacts and torsional strain.
Results
ANALYSIS OF PSA-ACT COMPLEX
An immunoassay, shown in Fig. 3, that is specific for the
PSA-ACT complex was developed by using mnAbs B80 or B87
imnmobilized on microtiter plates and rabbit anti-ACT poly-
clonal antibody. Goat anti-rabbit antibody conjugated with
HRPO was used as the signal-generating antibody. Free PSA or
ACT was not detected in this assay format. Goat anti-rabbit-
HRPO conjugate and rabbit anti-ACT antibody did not bind toimmobilized mAbs B80 or B87 (data not shown). Twofold
dilutions of PSA-ACT from 500 [Ig/L PSA were made (the
concentration of analyte is expressed in [Lg/L coinpiexed PSA).The dose-response curves were linear up to 50 [Ig/L and
saturated at -500 [Ig/L PSA-ACT complex. As little as 20 [Ig/L
of complexed PSA could be detected in this unoptimized assay.
Because both antibodies recognize the complex, we can con-
clude that both epitopes are not on or near the region that binds
to ACT. Another experiment directly compared a fixed sample
of PSA with and without ACT in ratios of 1:0.5, 1:1, and 1:5.
Here again no differences were observed in the PSA values
obtained, further confirming the PSA specificity of the mAbs.
0 100 200 300 400 500 600
Antigen (pg/L)
0CO CO CCC’)) CD C’) 0 F.. CO CO CCC’)))’) C, OF.. .- CO CO CCC’)) CD C’) OF.. COO CCC’))
CCC CCC C’) ‘ CO CO CON F- CO 0)0)0 CCC CCC C’) CO CO CD F.. COO) 0)0,- CCC C’) C’)CCC CCC CCC CCC CC) C’)
Peptlde number
Clinical Chemist-ty 42, No. 12, 1996 1965
Fig. 3. Detection of PSA-ACTcomplex by B80 and 687 in a sandwichformat immunoassay.In this assay, mAb B80 or B87 was used to capture PSA or PSA-ACTcomplex.Rabbit anti-ACT serum was used to detect any immobilized PSA-ACTcomplex.Both B80 and B87 were able to bind to PSA when complexed to ACT. Free PSAor ACT will not be detected in this assay. The difference in sensitivity of thisassay to PSA and PSA-ACTcomplex is due to differences in reagents and assayformat. The PSA assay involves two mAbs. whereas this assay involved rabbitanti-ACT polysera and anti-rabbit polysera to detect the complex. The concentra-tion of complex is expressed as g/L of PSA. The assay was performed asdescribed in Materials and Methods.
EPITOPE SCANNING WITH PSA-DERIVED HEXAPEPTIDES
To further identify the PSA epitopes bound by the two mAbs,
we synthesized all the overlapping hexapeptides derived fromthe linear amino acid sequence on PSA. Fig. 4 shows the results
of the screening of PSA sequence-derived hexapeptides with
bsmAb B8OXHRPO. The PSA peptide number represents theposition of the first amino acid in the hexapeptide relative to the
PSA amino acid sequence. The data shown was not background
subtracted and illustrates the low background signal from thehexapeptides, which do not bind to the bsmAb. In contrast, themonospecific B80 antibody binding measured by a goat anti-
mnouse-HRPO or biotinyl B80 along with streptavidin-HRPOgave high backgrounds and numerous peptides identified as
(false) positives due to the several complex secondary polyclonalreagents used. Four potential epitopes, exhibiting strong bsmAb
binding, were identified by this method: peptide 2 1-27
(RGRAVC), peptide 30-36 (\TLVHPQ), peptide 53-59 (RH-SLFH), and peptide 195-20 1 (PLVCNG). A few other peptidesshow moderate reactivity. No significant binding to PSA-
derived hexapeptides was seen for B87 in this assay.
AFFINITY SELECTION OF B80- AND B87-SPECIFIC PEPTIDES
FROM A PHAGE PEPTIDE DISPLAY LIBRARY
A phage decapeptide display library [32] was used (4.4 X 10”particles in round 1) to identify peptides that would specifically
bind to B80 or B87 and mnimic PSA epitopes. The Sepharose
beads with immobilized antibody were used for the affinityselection, and the elution conditions were the same as those used
for the affinity purification of PSA. This column, with 133 pmol
of immobilized antibody, was able to bind 40 pmol of PSAwithout saturating (molar ratio of antibody to PSA of 3.3 to 1).
The molar ratio of antibody to phage was -70 to 1. The phagewas eluted by incubating resin-bound phage with PSA and acid
and separated by centrifugation, providing a yield of 3.1 X i#{248}
and 5 x l0 particles, respectively. The molar ratio of the
immobilized antibody to the PSA used to elute the phage was-2.7 to 1. Functionally, this ratio would be much closer,since
not all antibody combining sites would be available for PSAbinding because CNBr-based coupling is a random linkage of
macromolecules to the solid phase. In addition, the affinity of
the mAbs for PSA is high (i.e., Kd -2 X 10m0 molJL). Themolar ratio of PSA used to elute phage was 25 to 1. The ligand
eluate was then amplified and reselected (2 X l0 particles as
1.2
O.8C’)0
0.6C
20.4
0.2
Fig. 4. Epitope scanning: binding of bsmAb B8OxHRPO to PSA amino acid sequence-derived hexapeptides.Hexapeptides representing the entire PSA amino acid sequence were synthesized on derivatized pins. This histogram shows several PSA.derived peptides that boundB80 antibody.The assaywas performedas described in Materials and Methods.
1966 Jette et al.: PSA epitope mapping
‘)Frequency of appearance of each sequence from among those sequenced.
Underlined sequences are consensus sequences; these indicate motifs that the selected sequences have in common.
input in round 2) with ligand and acid to generate 2.3 X iO and7 X 102 particles, respectively. We chose to elute the phage
particles from immobilized antibody with ligand (PSA) and acidto selectfor PSA-specific sequences [32]. After the secondround, the yield increased by 140-fold for the ligand elution,
whereas a threefold increase was seen with the acid elution,
suggesting that antibody-binding phage had been selected. The
ligand eluates gave the highest enrichment, indicating that acideluate was less specific. Enrichment was calculated as the total
yield of phage from the affinityselection elution from round 2divided by total yield of phage from the affinity selection elution
from round I.
To selectsequences that were specificto B80 or B87, an
immunoblotting assay was performed on nitrocellulose lifts of
colonies of cells infected with affinity-selected phage. The
fraction obtained by eluting with acid in both rounds ofselection did not yield any antibody-specific phage clones.B80-positive clones were seen for both eluants with ligand in the
first round. B87-positive clones were only found in the eluant,which was eluted with acid in the first round and ligand in the
second round.
IMMUNOSCREENING OF PHAGE DISPLAYING B80- AND B87-
SPECIFIC PEPTIDES IDENTIFIED BY AFFINITY SELECTION
To identify the phage specifically binding to B80 and to B87, anin situ immunoassay was performned. Phage eluted from the
affinity beads were grown in E. co/i as individual colonies on agar
plates. A nitrocellulose filter was used to lift the phage-infected
colonies onto a solid support [32]. After washing, phage particlesbound to the membrane were screened for binding to the twomAbs in a standard immunoblot assay. Two sets of plates and
filters were prepared; one set was screened with B80 and the
other with B87. Table 2 shows the number of positive clones foreach eluate. Colonies were selected for DNA sequencing on thebasis of the intensity of the immunoblotting results. Screeningwith B87 revealed 24% positive colonies with the phage that
were eluted first with ligand and second with acid. The other
phage eluants did not bind to B87.
The resulting peptide insert sequences are shown in Table 3along with the frequency of appearance of each sequence from
among those sequenced. Although it is tempting to speculate
that higher frequency of a particular sequence is indicative ofpreferential binding relative to other sequences, such a conclu-
sion may be premature and beset with problems of genetically
identical siblings picked randomly in the screening step. In all,66 clones were sequenced. Many gave the identical DNA
sequence, indicating that they were copies of the same clone.
Five unique sequences were identified for B80 and eight for B87.
Consensus sequences in the Table are underlined and indicate
motifs that the selected sequences have in common and requirefurther study that they are equivalent to binding motifs. The
most interesting finding was that one consensus sequence wasfound to be homologous to a linear sequence on PSA, peptide
51-55 (-LGRHS-). This sequence closely matches the B80-
specific peptide sequence 53-59 (-GRHSLFH-) identified by
epitope scanning (Fig. 4). None of the B87-specific sequences
identified by this method is homologous to linear sequences
found in PSA. These sequences represent mimotopes, peptidesequences that mimic the natural epitope and are able to bind
specifically to the antibody paratope.
The only criteria used to assess homology was sequencealignment performed both manually and by the SEQSEE
Eluate
Table 2. Number of positive clones ideSelection procedure
ntlfled by lmmunoblottlng of phage-infeB87.posltive colonies
cted colonies.B80.positlve colonies
Round 1 Round 2 no./totai % no./total %
1 Ligand Ligand 0/1500 0 49/1500 3
2 Ligand Acid 0/1500 0 28/1508 23 Acid Ligand 238/983 24 7/209 3
4 Acid Acid 0/1500 0 0 0
‘)Four eluat es are generated after two rounds of panning.
Table 3. B80- and B87-specific peptide Insert sequences Identified by affinity selection of a bacteriophage peptide displaylibrary.
B80 sequences
WLGRPSRSGG (3)8
TLGRRSRPGG(2)TLGRPSGLVG(1)ANHTRGRPLV (3)
WGFDFGFGSS(2)SCLFYNTVCV (3)
NEWSDEENGQ(2)
B87 sequencesSVWGA.ASSPW(3)SRWVRRSSGW(4)SRWSRSRNPR (4)SQWVRRSSGT (4)
RPWPRRSSGW(2)SQWRRSSPW (5)SQWVRRSSG(5)
RFRQRATIAF (1)
Cross-reactive sequences
AFH1TGRIAA (3)
DVRAFHWWGR (5)
ADEAFHTVGR (4)
PRRRFHTTGA (3)
DVPSFHWWGR(3)LVHAFHUGA (3)
SVWGMSSPW (1)
PSA sequence45-60
NKSVILLGRHSLFHPE
ClinicalChernistiy42, No. 12, 1996 1967
software[33].Any homologous pattern less than three amino
acids was considered insignificant. Multiple alignments wereperformed with the inputsequences by usingSEQSEE with the
rbo.wtmatrixwith gap penalties of 10 and extension penalties of2. In the caseof the B80 bindingpeptides,those sequences that
clusteredtogether as being >40% identical(i.e.,the top four
sequences in Table 3) were aligned separatelywith SEQSEE.
Those residuesdisplaying>70% sequence conservationfrom
thissetwere used to form a “consensus”sequence.Thus forthe
B80 set,theconsensussequence would be TLGRPS***G (where
* isany amino acid).In the B87 set,a 70% sequence identity
thresholdwas alsoused.These criteriaidentifiedthe consensus
sequence S*W*RRSS. For the sake of simplification, only
conserved contiguous residues were underlined in Table 3. Aseries of alignments was performed with the eight sequences in
the B87 column (Table 3) against the PSA sequence (PIRaccession A26757) with SEQSEE. The same default scoring
matrix and gap penalties were used. The only sequence that
yielded any reasonable score was at the N terminus(WECEKHSQPW) of PSA. This particular sequence still re-
quired the introduction of two gaps and only two of the most
highly conserved residues were matched. By any criteria, this is
a poor match. Typically over a stretch of 10 residues, one shouldexpect to find better than 60% sequence identity (with no gaps
or breaks) if the match is significant. The nonspecific or
cross-reactive sequences (Table 3) appear to be reactive to both
mouse antibodies B80 and B87 and may be motifs capable of
binding the constant domains of IgG 1. This, however, requires
further study.
COMPUTER MODEL OF PSA
A structural model of the molecule, if not available from x-ray
crystallography, can be generated by using proteins of knownstructure that have a high degree of homology with the antigen.
The model can assist in locating highly exposed surface regions
and illustrate the spatial relations between various binding or
catalytic sites and antibody epitopes.Figure 5 shows the computer-generated homology model of
PSA. The catalytic triad for this serine protease-like molecule
(His-41, Asp-96, and Ser-71) is shown in white and the locationof the ACT binding site, based on what is known about the
binding of ACT to chymouypsin, is indicated by the shadedresidues. The B80 epitope identified by hexapeptide scanningand phage affinity selection, residues 53-60, is indicated inyellow. It is seen to be on an exposed region of the molecule wellaway from the ACT binding site. An epitope for B87 was notidentified in this study and is likely in an exposed region that is
not masked by the binding of ACT or B80 to PSA.
Computer models of PSA have been described by Vijinen
[34] and Bridon and Dowell [35]. The three-dimensional struc-ture of PSA and glandular kallikrein were modeled on the basis
of pKl. Loops unique to PSA were modeled by searching froma database and the structure was refined by energy minimization
and molecular dynamics. The resulting model closely resemblesthe PSA model generated in this study (Fig. 5). From the
location of the binding site of ACT it is apparent that most ofthe molecule is still exposed and available for binding to
Fig. 5.MolecularmodelingofPSA: The catalytic residues are shown inwhite and theproposedB80 epitope in yellow.The modelwas created as described in Materials and Methods.
antibodies when ACT is complexed to PSA. The B80 epitope(shown in yellow in Fig. 5), located in this study, is on a highly
exposed region near the N terminus, well away from the ACTbinding site. The exact location of the B87 epitope was not
determined but the possible epitopes could be limited to ex-posed regions that are unique to PSA and that do not overlap
with the B80 epitope or the ACT binding site.
Discuss#{226}onIn this study we attempt to addressthe issueof immunospeci-
ficity of anti-PSA antibodies at the molecular level by charac-terizing the epitopes recognized by two new mAbs. At least fivePSA isomers have been identified with pls ranging from 6.8 to
7.5. This heterogeneity is believed to be caused by differing
sialic acid content of the carbohydrate side chains and not by
changes in the amino acid sequence [9]. All of this variabilitymeans that antibodies directed against specific epitopes may
recognize some forms of PSA and not others. There may beepitopes on PSA that are especially sensitive to the conforma-
tional changes that occur when the enzyme is converted fromprecursor to zymogen to enzyme to inactive enzyme. Antibodies
directed against conformationally sensitive epitopes may only
recognize one of these forms of PSA.Two anti-PSA mAbs designated B80 and B87 were used in
this study. The B80 hybridoma cell line was fused in ourlaboratory with an anti-HRPO cell line to produce a hybrid
hybridoma or quadroma secreting a bispecific mAb with one
binding site directed against PSA and the other against HRPO[31]. This useful reagent eliminates the need for biotinylated or
secondary antibody-based detection methods and provides uswith a probe potentially having the highest specific activity ofHRPO-generated signal. Further, this bispecific mAb probeeliminates the high background often created by the secondary
polyclonal antibody- enzyme conjugates used in conventionalimmunoassays.
1968 Jette et al.: PSA epitope mapping
B80 and B87 were used to measure PSA in a sandwichimmunoassay, indicating that they recognize two distinct and
nonoverlapping epitopes. The PSA used to calibrate this assay
was obtained commercially as 99% pure protein in BSA buffer
to ensure that PSA-ACT complex was not present in this
calibrator. The ability of these antibodies to bind to PSAsimultaneously indicates that the epitopes are nonoverlapping
and far enough apart to avoid steric hindrance. Both of these
antibodies fail to form a homosandwich, indicating that each
antibody recognizes one unique and nonrepeating epitope. This
result is not unexpected considering the relatively small size of
PSA. Another recent study [36] also demonstrates by Western
blot analysis that the mAb B80 binds most consistently (com-
pared with other commercially available monoclonals studied)
not only to freePSA and PSA-ACT but also to PSA complexed
with AMG.
The specificity of the antibodies was determined by measur-ing the degree of cross-reactivity with pKl, which shows a
59.6% amino acid sequence homology with PSA. The fact thatneither B80 nor B87 cross-reacts with this related protease
shows that both antibodies recognize sequences on the molecule
that are not common to pKl and PSA. Since we can also assume
that the epitope resides on an exposed surface structure and is
not hidden in the molecule, the number of possible epitopes is
greatly reduced. Further studies on cross-reactivity with hK2
(84% homology to PSA) is needed to establish the unique
specificity of these mAbs.Sequential or linear epitopes are continuous strands of
residues present on the surface of a protein. Discontinuous or
nonlinear epitopes are sequences brought into proximity on the
surface of the molecule. Antibodies to discontinuous epitopesmay react weakly to subregions of the epitope made up of a few
residues in a linear sequence. Linear epitopes can be identified
and located by epitope scanning, a technique first introduced by
Geyson et al. [37]. It is based on the principle that linear
epitopes can be mimicked by peptides with as few as six residues.
Some of these peptides represent actual sequences found on the
antigen and indicate the location of the epitope on the protein
molecule. Others mimic the conformation and binding charac-
teristics of the epitope and can bind specifically to the antibody.
These peptides are referred to as mimotopes. Hexapeptides
representing all possible overlapping amino acid sequences of
PSA were synthesized on a solid-phase pin and tested for
binding to B80 and B87 in an ELISA format. The hexapeptidescan identified four possible locations of the B80 epitope. Two
of these peptides at residues 30-36 and 195-201 are found in the
interior of the molecule (on the basis of a computer-generated
model of PSA [34]), and are hence not likely candidates for the
epitope. The remaining two sequences at 21-27 and 5 3-59 are
adjacent to one another on the surface of the molecule. This
technique of epitope scanning, which can identify linear
epitopes that can be defined by a short linear peptide, failed tolocate the B87 epitope. It is likely that B87 recognizes a
conformational epitope that cannot be mimicked by a short
peptide based on the linear sequence of PSA. It is also possiblethat the epitope for B87 may be a linear epitope that is >6
residues. A short peptide representing a part of this epitope may
not be able to bind to the antibody paratope. Further, differ-
ences in conformation caused by interactions with neighboring
residues that are not directly involved in binding may be critical
to binding. A short peptide will not be able to mimic this
conformation and thus may not bind to the antibody.
Bacteriophage peptide display libraries can be used to affin-
ity-select peptides that specifically bind to a given antigen or
antibody [38-40]. The major advantage of using recombinant
peptide libraries is that a huge diversity of conformational
structures can be created and screened within a single library. By
this approach, a bacteriophage display library [32] was used to
identify sequences that bind specifically to B80 or B87.Among the phage sequences identified (Table 3), one con-
sensus sequence is homologous to a linear sequence seen on PSA
and identified as a possible epitope by epitope scanning. The
fact that two different techniques independently identified the
same region on PSA as the B80 epitope is strong evidence that
the epitope involves residues 51-54 (GRHS). The other anti-
body-specific sequences (Table 3) may represent mimotopes,peptidesthatmimic the conformation of theepitope.The other
remaining sequences listedin the third column of Table 3 are
peptides that were cross-reactivewith B80 or B87. This con-sensus sequence has been observed in other unrelated affinity
selection experiments and may represent cross-reactive binding
to constant regions of antibodies. The other potential PSA
mimotopes identified by this method are currently being eval-
uated.
In conclusion, the epitopes of two high-affinity anti-PSA mAbs,B80 and B87, were studied and well characterized. They bind
both free PSA and PSA complexed with ACT, and do not
cross-react with pKl or hK2. To locate the epitopes, overlap-
ping hexapeptides representing the entire PSA amino acid
sequence were synthesized and screened in an immunoassay for
binding with the two mAbs. B80 showed specific binding to
several peptides. B87 did not show binding to a specific peptide,
suggesting that the B87 epitope may be nonlinear. A bacterio-
phage peptide display library was used to select for peptide
sequences that bind specifically to B80 and B87. Several con-
sensus sequences were identified with this technique. One
peptide sequence was homologous to a linear sequence on PSA,
which was also identified as a possible B80 epitope by PSA
hexapeptide scanning.
MRS. acknowledges Biomira Inc. and the Medical Research
Council of Canada (MRC) for support of a University-IndustryChair, and the Natural Sciences and Engineering Research
Council of Canada (NSERC) for a strategic grant and ACB for
a group grant.
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