May 16, 1991 - P. Steiner,'. Bryant. M. Moore ..... by David. Sane,. Duke. University. Med- ical Center. Prothrombin and activation ... 2 mmol/L. Anti -p rot hrombi.
CLIN. CHEM. 39/4, 583-591
Monoclonal Quantitative
(1993)
Antibodies Specific for Prothrombin Fragment Enzyme-Linked Immunosorbent Assay
Marcie J’ Hurstmg,’ Bryan Szewczyk,’ Maria L. Bell,”4
Butman,2
T.
Jerald
and Frederick
P. Steiner,’
98%±13%; no interterence from lipemia, hemolysis, icterus, or thrombolytic agents; 0.08 nmol/L sensitivity; and mean intra- and interassay imprecision (three lots) 95%, >95%, and >99%, respectively,
Mono-Q)
as determined by SDS-PAGE. Purified proteins were stored at -70 #{176}C in the presence of benzamidine, 2 mmol/L. Anti -p rot hrombi n(Ca2 ) antibodies. Antibodies specific for the calcium-dependent conformation of prothrombin (i.e., Fl region) (10) were isolated from rabbit anti-prothrombin antisera by conformation perturbation affinity chromatography (13). Conjugates. Conjugates of synthetic peptides with ovalbumin and HRP and of anti-prothrombin(Ca21 antibodies with HRP were prepared by using the cross-
All chemicals were reagent unless otherwise noted, purchased from Sigma Chemical Co. (St. Louis, MO). N-Succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) was obtained from Pharmacia (Piscataway, NJ). Synthetic peptides were purchased from Cambridge Research Biochemicals, Cambridge, UK (peptides PF2, des-RPF2, XPF2, and PF2.14; see Table 1), or Multiple Peptide Systems, San Diego, CA. Type V ovalbumin, horseradish peroxiChemical.s/biochemicals. grade or better and,
Acid Sequences
of Synthetic
IN). were Med-
ical Center.
Materials
1. Amino
Oxyuraneous snake venom were purSigma. Purified human prothrombin and thrombin were purchased from Enzyme ReInc. (Indianapolis, IN) and purified human factor
X8 from Boehringer Mannheim (Indianapolis, Thrombolytic agents used in interference studies kindly provided by David Sane, Duke University
and Methods
Table
and
Peptides
and PartIal
of F1.2, F2, and Prothrombln
Sequences
No. of
amino acids
S.qu.nc.
Cemmsnts
Peptlde
PF2 des-RPF2 XPF2 PF2.14 PF2.K PF2.NH2 PF2.8 PF2.8
10(8 + 2) 9(7 10(8 14(12
+ 2) + 2) + 2)
8 8 8 6
PF2.5 PF2.4
5 4
PF2.3 IP
CGSDRAIEGR
Neoepitope
CGSDRAIEG
C-terminal
CGAIEGRTATS CGLDEDSDRAIEGR SD RAIEG K S D A A I E G R-amide SD RAIEG A RAI EG R AIEGR IEGR EGA
3 7(5
Native protein Fl .2 F2
271 116
Prothrombln
530
+
2)
CGOVDPR
...LDEDSDRAIEGR ...LDEDSDRAIEGR LDEDSDRAIEGRTATS..
CLINICAL
CHEMISTRY,
Vol. 39, No.4,
1993
C-terminus of Fl .2 Fl .2
Includes factor X cleavage site of prothrombln Longer form of PF2 Lysine substituted for C-terminal arginine of Fl .2 Amidated form of C-terminal arginine of Fl .2 Eight C-terminal amino acids of Fl .2 Six C-terminal amino acids of Fl .2 Five C-terminal amino acids of Fl .2 Four C-terminal amino acids of Fl .2 Three C-terminal amino acids of Fl .2
Irrelevant peptide Prothrombin fragment 1.2 C-terminal domain of Fl .2 Native
protein
Allsyntheticpeptides have a free carbox group at the C-terminus except for PF2.NH2, which has an amidated the amino acids C and G at their N-terminus forconjugationputposes only. 584
on
des-arginine
C-terminus
(-CONH2).
Five peptides has
linking agent SPDP Thiolation of synthetic
according peptides
the presence of N-terminal gates containing synthetic ‘tpeptide-carrier Preparation BALB/c
to Carlsson et al. (14). not required, given
was cysteine peptides
residues. are referred
Conjuto as
protein.”
and Screening
immunized with PF2-ovalbumin modifications of the procedure of Cianfriglia et al. (15). Briefly, mice were injected intraperitoneally on day 1 with 50 pg of PF2-ovalbuimn in complete Freund’s adjuvant, then rested for 21 days. On day 22, the mice were boosted intraperitoneally with 50 pg of PF2-.ovalbumin in incomplete Freund’s adjuvant. Final boosts were administered on days 25, 26, and 27 with 5 pg of immunoconjugate in phosphate-buffered saline, pH 7.2, delivered both intraperitoneally and intravenously. On day 28, the mice were killed, their spleens were removed, and splenocytes were isolated for
conjugate
mice
of Hybridomas
were
in slight
fusion. Splenocytes were fused to a nonsecretor cell line P3X63Ag8.653 (ATCC no. CRL 1580) (16) and plated into Iscove’s Modified Dulbecco’s Medium containing hypoxanthine, aminopterin, and thymidune (HAT) with
fetal bovine serum and either equine serum or media supplement (Nutridoma; Boehringer Mannheim). Cultures exhibiting hybridoma growth in HAT medium were screened for production of anti-F1.2 antibodies. For this purpose, hybridoma supernates were incubated
(60
miii, 37#{176}C)in Immulon II micro-ELISA wells Laboratories, Chantilly, VA) coated with After a wash, the wells were incubated (60 miii,
(Dynatech F1.2.
37#{176}C) with HRP-labeled
goat antibodies to mouse immunoglobulin (HyClone Laboratories, Logan, UT). After washing, HRP activity was determined by using tetramethylbenzidine (TMB)/hydrogen peroxide (Kirkegaard and Perry Laboratories, Gaithersburg, MD) as substrate, 1 mol/L sulfuric acid as quench reagent, and 30 mm for color development.
Absorbance
at 450
nm
was measured
with a microplate reader (Vmax; Molecular Devices, Menlo Park, CA). Positive hybridomas were expanded and retested for antibody specificity by using microELISA wells coated with F1.2, PF2-ovalbwnun, ovalbu-
mm,
or prothrombin.
immunodiffusion
Isotype (The
Binding
was determined by double Site, San Diego, CA) of
the hybridoma supernates. Hybridomas selected on the basis of specificity and isotype were cloned by two rounds of limiting dilution into aminopterun-free, L929 murune fibroblast-conditioned
hybridoma culture medium. Stable hybridoma propagated as ascites tumors in CD2 Fl mice (BALB/c x DBA Fl hybrid). Monoclonal antibody specificity in 1000-fold-diluted ascites fluid was determined by using antigen-coated micro-ELISA wells as described above and, as diluent, PBS plus fish gelatin (Norland Products, New Brunswick, NJ), 30 g/L.
clones
were
Purification
and Characterization
of Monoclonal
Antibodies
Monoclonal antibodies were purified from ascites fluid by Protein A-Sepharose affinity chromatography (Pharmacia). Antibody concentrations were determined from
the absorbance at 280 nm with use of an absorptivity of 1.4 L g1 ‘cm’ for IgG. Isoelectric points were established by isoelectric focusing (Pharmacia Phast-Gel system). Specificity of the purified monoclonal antibodies was determined by the above-described assay but with Griener micro-ELISA plates (Organon Teknika Corp.), HRP-labeled goat anti-mouse IgG (heavy + light chains; Boehringer Mannheim), and 10 mm for color development. Reactivity was defined as an absorbance at 450 nm greater than three times the absorbance of the background when 0.5 pg of antibody was tested. The specificity of monoclonal antibody 5-3B was confirmed by Western blot. Purified monoclonal antibodies (5-3B anti-Fl.2 or an anti-Fl positive control) were incubated (5 mg/L, 60 miii, 37 #{176}C) with Western blot membranes containing purified antigens (prothrombin, thrombin, P1.2, Fl, and F2) transferred from an SDSPAGE gel (8%-16% gradient acrylamide). After a wash, the bound antibodies were made visible as described above for the ELISA but with use of HRP-labeled goat antibodies to mouse immunoglobulin (HyClone Laboratories) and TMB/membrane peroxidase substrate (Kirkegaard and Perry Laboratories). The molecular masses of the stained bands were determined by using prestained molecular mass markers (Rainbow Markers, range 14.3-200 kDa; Aniersham International, Amersham, UK). The ability of a monoclonal antibody to capture solution-phase P1.2 was evaluated by incubating (60 mm, 37#{176}C) purified P1.2 in antibody-coated Griener microELISA wells, washing the wells, and incubating (60 mm, 37#{176}C)HRP-labeled anti-prothrombun(Ca2) antibodies in the wells. After another wash, HRP activity was determined with 10 miii for color development. The epitope specificity of monoclonal antibody 5-3B was evaluated by competitive inhibition assay. PF2.14HRP (50 zL of 10 nmoJJL reagent) and an equal volume of various concentrations of purified P1.2 or synthetic peptide were incubated (75 miii, 37#{176}C)in 5-3B-coated Griener micro-ELISA wells. After awash, HRP activity was determined, which reflected the extent of PP2.14HRP binding in the presence (B) or absence (B0) of competing antigen. Quantitative
ELISA
of Fl .2 in Plasma
Six calibrators containing 0-10 nmol of F1.2 per liter and two controls containing low (Level I) and high (Level II) concentrations of F1.2 were prepared by adding purified P1.2 to BaSO4-adsorbed, P1.2-depleted human plasma. For the assay, we incubated (60 mm, 22-25 #{176}C) plasma sample, calibrator, or control in dupli-
cate in monoclonal antibody 5-3B-coated micro-ELISA wells. The wells were washed by first aspirating their contents and then performing four cycles of filling! aspirating with a surfactant-containing wash solution. After the wash, HRP-labeled anti-prothrombin(Ca2) antibodies were incubated (60 mm, 22-25 #{176}C) in the presence of calcium in the wells. After another fourcycle wash, HRP activity was measured by using the CLINICAL
CHEMISTRY,
Vol. 39, No.4,
1993
585
acid indicator system as described above and a lO-min color development. Mean absorbance values at 450 nm from calibrators or samples were used, respectively, for constructing the calibration curve or for determining the unknown P1.2 TMB/peroxide/sulfuric
concentrations. Plasma
Samples
Blood evacuated units/mL
was
for Fl .2 Measurement
collected by routine venipuncture into tubes containing lithium heparin (14.3 USP blood) and then centrifuged (l000xg, 15 miii, 4#{176}C) to obtain plasma. Plasma was diluted (9/1, by vol) with an EDTA-containing anticoagulant formulated for use with heparinized plasma (Sample Treatment Rea-
gent;
Organon
Teknika Corp.), and then either assayed or stored at -20 #{176}C. Dilution with Sample Reagent was required both to avoid artifactual changes in Fl.2 concentrations during storage and to ensure compatibility (i.e., minimal matrix effects) between assay calibrators and heparinized samples. immediately Treatment
Resutts
Monoclonal
Antibodies
of stable clones with anti -F12 activity. A of synthetic peptide PF2 and ovalbuinin (PF2-ovalbumin) was used as the immunogen for obtaining P1.2-specific, murine monoclonal antibodies. The sequences of PP2 and other peptides used for antibody screening and characterization as well as the partial sequences of human P1.2, P2, and prothrombun (8) are shown in Table 1. The PF2 sequence has the same eight C-terminal amino acids as F1.2, plus two additional amino acids (cysteine and glycine) at the N-terminus for conjugation purposes. PF2-ovalbumin was prepared via conjugation of the cysteine thiol group of the peptide to ensure a free carboxyl group at the PF2 Preparation
conjugate
by the
hybridomas. All monoclonal reactivity proffles against the antigens 2). Each antibody recognized native P1.2, P2, and the ovalbumin conjugate of the synthetic peptide similar to the P1.2 C-terminus (PF2ovalbuinin). Each antibody failed to recognize the immunogen’s carrier protein (ovalbumin), prothrombun, and prothrombin-derived proteins not containing the C-terminal SDRAIEGR sequence (Fl and thrombin). Also, an ovalbuinin conjugate with the des-argunine form of PF2 (desRPF2-ovalbumin) was not recognized, indicating that the presence of a C-terminal argimne was critical for antibody binding. After purification from ascites, the monoclonal antibodies were further characterized in terms of physical properties and, again, specificity to confirm their reactivities (Table 2). Five of the six monoclonal antibodies were of the IgG1 subclass and had isoelectric points (p1) between 6.0 and 7.0. One antibody, 5-1OH, was of the IgG2 subclass with p1 >8.0. Isoelectric focusing patterns for each were consistent with the presence of monoclonal, not polyclonal, antibodies. As did antibodies in ascites fluid, all six purified antibodies bound specifically to P1.2, P2, and PF2ovalbumun (Table 2). No specific antibody binding was observed to Fl, prothrombin, thrombin, or the pyridyldisulfidated form of ovalbumin generated during SPDPbased coupling. Also, no specific binding was observed to conjugates of ovalbunun with synthetic peptides similar to either the F1.2 C-terminus without arginine (desRPP2); the prothrombin R271-T272 region, the cleavage site for factor Xa (XPF2); or an irrelevant amino acid sequence (IP). Background absorbance values were higher with antibody 5-1OH than with the other antibodies, indicating increased nonspecific interactions of this antibody with the micro-ELISA wells and (or) culture
antibodies
cloned
six
showed similar tested (Figure
C-terminus.
The schedule for immunizing mice with PP2-ovalbumiii was critical for generating anti-F1.2 hybridomas. Five attempts at obtaining the desired hybridomas by using either a conventional immunization schedule or the standard, 14-day procedure of Cianfriglia et al. (15) produced relevant polyclonal responses but no specific hybridomas. Modification of the latter procedure to include a 21-day rest period after priming resulted in generation of the numerous anti-P1.2 hybridomas described below. Screening assays of 848 hybridomas obtained after immunization and fusion steps revealed 44 with antiP1.2 activity. After expansion, 8 of these 44 hybridomas expressed supernatant reactivity with F1.2 and PF2ovalbumin, but not with ovalbumin or prothrombin, and demonstrated an IgG isotype. After cloning, six of the eight hybridomas demonstrated acceptable stability and were propagated as ascites tumors in mice. These hybridomas and their associated antibodies were designated 6-9A, 8-11H, 9-lA, 2-7C, 5-3B, and 5-1OH. Characterization
Monoclonal characterized 586
CLINICAL
of anti-Fl 2 expressed
antibodies to confirm
CHEMISTRY,
monoclonal in ascites
the specificity
antibodies. fluid were
demonstrated
Vol. 39, No. 4, 1993
in
-
P,o*O,,b1.
TI.,b4n
EJ
FI.
Pt
P1
PPS-OVA
.PPt-0VA
OVA
a E C
0 Ui ,
2 01
4-
0 S S
0
2-iC
5-3B
5-lOll
Monoclonal
Fig. 2. Reactivity profiles for monoclonal against various antigens
6-BA
B-I1H
9-lA
Antibody
antibodies in ascites fluid
Diluted ascites fluid was incubated with a specificantigen Immobilized on a micro-ELISA st,lpweli, and bound antibody was detected by using peroxidaselabeledanti-immunoglobulin antibodies, with peroxidase activitymeasured at 450 nm. OVA. ovalbumin; PF2-OVA, a synthetic peptide-ovalbumin conjugate emulating the Fl.2 C-terminal;desRPF2-OVA, a synthetic peptideovalbumln conjugate emulating the Fl .2 C-terminalbut missing arginine
Table 2. Reactivity
and isotypes
Profile
Anti-Fl
of Purified
.2 Monocionai
Proteins Manoclonal
Pyr-
antibody 5-9A
IgG1 IgG1 lgG1 lgG1 lgG1 ig%,
8-11H 9-lA 2-7C 5-3B 5-1OH Pyr-OVA, control
p1
pylidyldisulfidated
Prothrombln
Thrombin
PF2OVA
desRPF2-
XPF2-
IP-
OVA
OVA
OVA
Fl
6.0-6.6 6.4-6.8
+
-
+
-
-
#{247}
+
-
+
-
-
+
6.4-6.8
+
-
+
-
-
+
6.5-7.0
+
-
+
-
-
+
6.6-7.0
+
-
+
-
-
+
>8.0
+
+
-
-
+
ovalbumin
generated
during SPDP-based
crosslinking;
IP-OVA,
OVA
p.ptlds.-ovalbumln
Fl.2
-
F2
Antibodies
Synthetic
synthetic Irrelevant
conjugatss
-
±
-
conjugate
peptide
representing
negative-
antigen.
conjugate, possibly associated with the IgG2 subclass or basic p1. The specificity of monoclonal antibody 5-3B for the P2 domain on Fl.2 was confirmed by Western blot (Figure 3). This antibody bound to bands corresponding to Fl.2 (43 kDa) and F2 (16 kDa) but not prothrombin (72 kDa), thrombin (38 kDa), or Fl (28 kDa). A control antibody specific for the Fl domain stained bands in a Western blot corresponding to prothrombin, Fl .2, and Fl, but not those for thrombin or P2 (data not shown). We evaluated the ability of the monoclonal antibodies immobilized on the micro-ELISA plates to capture solution-phase P 1.2. The resulting P1.2 binding isotherms are shown in Figure 4. All six antibodies yielded reasonable dose-response curves for F1.2 >1 nmol/L. However, antibodies 5-1OH and 5-3B were superior for binding lower concentrations of P1.2. Because of the greater nonspecific interactions previously observed with 5-1OH, we selected antibody 5-3B for further evaluation and use in development of our quantitative sandwich ELISA for F1.2 in plasma. Epitope specificity of monoclonal antibody 5-3B. To determine the abilities of native P1.2 and various synthetic peptides to compete with an HRP-labeled, 14-
1.60 --
5-1OH
-#{232}--
5-3B
-#{149}-
9-lA
/
1.20
/
/
U #{149}#{149}#{149}O#{149}#{149} 8-11H
E
--
6-9A
-#{176}--
2-7C
C 0
0.80
I’) ,
0.40
0.00
0.01
01
1
10
nmol/L
[Fl.2] Fig. 4. Fl .2 binding isotherms
to
100
purified
anti-Fl .2 monoclonal
antibodies
Various concentrationsof purified Fl .2 were incubated with monoclonal antibody immobilized on a micro-EUSA well. Bound Fl.2 was detected by using
peroxidase-labeled
antI-prothrombln(Ca2)
antibodies,
Fl .2 N-terminal region measured
in the presence of calcium; peroxidase with a chromogenic indicatormonitored at 450 nm
peptide P1.2 (PF2.l4-HRP)
resembling the for binding to used a competitive ELISA format format. This avoided the need for with potentially variable reactivity amino-acid
Fig. 3. Western blot reactivity of anti-Fl .2 monoclonal
antibody
5-3B
Purified 5-3B was used in Western blot to immunostain protein bands transferred from an SDS-PAGE gel. Bound antibody was detected by using peroxidase-iabeled anti-immunoglobuiln antibodies. Specific reactivity was shown with F1.2 (lane 4) and F2 (lane 1) but not with prothrombln (lane 2),
thrombin (lane 3), or Fl (lane 5)
C-terminal immobilized
which bind the activity was
region of 5-3B, we
instead of a sandwich a detection antibody against the synthetic peptides tested. For the competing antigen, we used an HRP conjugate with PP2.14, instead of the shorter peptide PP2, to allow evaluation of the critical epitope size over a wider range of peptide lengths (see below). Both unlabeled PF2.l4 and P1.2 effectively competed with PP2.14-HRP on a molar basis (Figure 5), validating the assay and confirming the ability of antibody 5-3B to recognize both native Fl.2 and synthetic peptides resembling the C-terminus of P1.2. Figure 5 also shows the competitive binding isotherms obtained for PF2.14-HRP vs synthetic peptides CLINICAL
CHEMISTRY,
Vol. 39, No. 4, 1993
587
1.20
100 1.00 C .
0.80
C
m 0.60
80
0
cc 0.40 I-
0.20
60 U0
0.00 0.01
0.1
1
10
100
1000
10000
C 0 4-
Antigen
Concentration
(nmol/L)
.0
Fig. 5. Epitope specificity of monoclonal antibody 5-3B evaluated by competitive inhibition assay with a peroxidase-labeled, 14-aminoacid synthetic peptide similar to the Fl .2 C-terminus (PF2.14-HRP) Competitive binding isotherms were generated by incubating an equivoiume mixture of various concentrations of competing antigen and 10 nmol/L PF2.14-HRP with antibody 5-3B Immobiiized on a mlcro-ELISA well. Bound PF2.14-HRP (B) was detected with a peroxidase chromogenic indicator at 450 nm; B/B,, represents the fraction PF2.14-HRP bound in the presence of competing antigen (PF2.14, Fl .2, PF2.K, PF2.NH2, and des-RPF2). See Table 1 for sequences of synthetic peptides
similar to the F1.2 C-terminus with nine missing (des-RPF2), amidated
its
terminal
presence of any C-terminal amino acid with a free carboxyl group (e.g., des-RPF2) was not sufficient. Nor was the presence of a C-terminal arginine without a free carboxyl group (e.g., PF2.NH2) sufficient for recognition. The presence of a C-terminal positively charged amino acid with a free carboxyl group (e.g., PF2.K) was important but not sufficient. Optimum recognition occurred when a C-terminal arginine with a free carboxyl group
(e.g., PF2.14) was present. The relative abilities of synthetic peptides of differing lengths to compete with PF2.14-HRP for antibody 5-3B binding sites were evaluated to establish the critical epitope size. The sequence of each competing peptide was similar to the F1.2 C-terminus but varied in length from 3 to 14 amino acids (PF2.3, PF2.4, PF2.5, PF2.6, PF2.8, PF2.14, see Table 1). Maximum differences in percent binding inhibition were observed when using 10 .anolIL competing peptide (1000-fold the PP2.14-HRP concentration), and results obtained at this peptide concentration are shown as a function of peptide length in Figure 6. Minimal change in the percent inhibition of binding occurred when the competing peptide’s length increased from 3 to 6 or from 8 to 14 amino acids. However, the percent inhibition of binding approximately doubled when the competing peptide’s length increased from six to eight amino acids, indicating a critical epitope size within that range.
The
F1.2 588
Performance two-stage,
uses
quantitative
monoclonal
antibody
direct
5-3B
CLINICAL CHEMISTRY, Vol. 39, No. 4,
of plasma immobilized on
ELISA
1993
40
C
20 0
In
argi-
(PF2.NH2), or replaced by lysine (PF2.K). Peptides des-RPP2 and PF2.NH2 failed to compete with PF2.14-HRP, and peptide PF2.K competed only at high concentrations. For monoclonal antibody 5-3B to recognize an epitope, the
Assay
-C
Fig. 6. Critical monoclonal
2
4
6
#
of
Amino
Competing
epitope size required antibody 5-3B
8
10
12
14
16
Acids Peptide
for optimal
recognition
by
binding isotherms for PF2.14-HRP to antibody 5-3B were generated as described in Fig. 4 with the competing antigens being synthetic peptides that resembled the Fl .2 C-terminus. Percent inhibition of PF2.14HRP binding measured at 10 Iimot/L competing antigen is shown as a function of the number of amino acids in the competing peptide Competitive
micro-ELISA stripwells to capture F1.2 and detects captured P1.2 with HRP-labeled anti-prothrombin(Ca21. In the first stage, immobilized antibody binds F1.2 from sample, calibrator, or control via the P1.2 C-terminus; then, unbound proteins, including prothrombin, are removed by thorough washing. In the second stage, HRP-labeled anti-prothrombin(Ca2) antibodies specific for the calcium-dependent conformer of the N-terminal region of F1.2 and of prothrombin are added to the wells in the presence of calcium. The conjugate binds captured P1.2 via its N-terminal region. After another washing to remove unbound conjugate, a chromogenic indicator system is added and HRP activity is measured at 450 urn. Figure 7 is a typical calibration curve generated by plotting absorbance at 450 urn vs the logarithm of the F1.2 concentration of each calibrator. As with this example, a four-parameter data fit of the calibration curve generally yielded a correlation coefficient 0.99. Analytical recove1y. A mean recovery of 98% (SD = 13%, n = 23) was obtained when purified F1.2, from 0.5 to 18 nmoIJL, was added to plasma samples from 11 healthy donors. Samples containing >10 nrnol/L P1.2 were diluted with the zero PL2 calibrator to give results within the assay’s working range. Parallelism. P1.2 concentrations were measured in serial dilutions (n =4) of three plasma samples containing >10 nmol/L PL2. Dilutions were appropriate to
1.80
Table 3. ReproducibilIty L.vel
1.35 Kit lot
of Fl .2 ELISA
I control
Fl.2,
CV,
nmol/L
%
Level II control
n
CV,
F12, nmol/L
%
Intraassay 0.90
A
0.45
B C Mean
0.49 0.55 0.57
3.3 10.9 14.8
20
6.49
7.3
20
27
6.55
6.93
12.6 14.7 11.5
15 21
6.97
7.6
20
7.43
8.3
14
6.71
6.7 7-5
27
6
9-7 Inferassay
0.00 0.01
0.1
1
F1.2
10
best-fit regression. For plotting is represented as 0.01 nmoVL on
P1.2
0.51 0.48
8.6 7.3 10.4 8.8
and 7.5%, respectively. reported for other analytes (2, 17). 8.8%
that
Clinical
ensure that unknown values fell working range (0-10 nmol/L F1.2), calibrator was used as diluent. calculated for undiluted samples the extent of dilution (CVs 10 nmol/L, consistent with competition for binding sites on immobilized antibody 5-3B in the assay. F1.2 concentrations were also measured in a threedonor plasma pool in the absence and presence of triglyceride 5.0 gIL, hemoglobin 1.0 gIL, bilirubin 200 mg/L, streptokinase or urokinase 500 kU/L, or tissue plasminogen activator 1000 kU/L. Percent recovery of P1.2 ranged between 92% and 111%, indicating no significant assay interference by these substances at the concentrations tested. Sensitivity. For six different lots of reagents, the minimum detection limit ranged between 0.04 and 0.08 nmol/L. These values were based on the lowest F12 value distinguishable from the zero F1.2 calibrator (i.e., the P1.2 value corresponding to the mean A plus 2 SD for the 0 nmol/L calibrator; n = 16). Reproducibility. Intraand interassay imprecision were established for three lots of reagents by assaying the low (Level I) and high (Level II) P1.2 controls (Table 3). The number of runs performed differed between the three lots only because of limits in reagent availability. For these lots, the mean intraassay CVs for the Level I and II controls were 9.7% and 11.5%, respectively. The mean interassay CVs for the Level I and II controls were
Imprecision
sandwich
was ELISAs
similar to of plasma
Application
For a healthy population younger than age 44 years (n = 268), the geometric mean concentration of plasma F1.2 measured with this P1.2 ELISA was 0.51 nmol/L (95% tolerance interval: 0.21-278 nmol/L), in general agreement with previously reported mean values determined by polyclonal antibody-based immunoassays (1, 2). Details of this study appear elsewhere in this issue (18).
Discussion The goals of this study, i.e., to obtain anti-F1.2 monoclonal antibodies and to develop a highly specific, monoclonal antibody-based F1.2 immunoassay, were achieved. Six monoclonal antibodies reactive with human plasma P1.2 but not with prothrombin were obtained and characterized. One of these monoclonal antibodies (5-3B) exhibited superior ability as a capture antibody and was successfully used with a calciumdependent antibody to the F1.2 N-terminus to demonstrate a specific, two-stage direct ELISA for P1.2 in human plasma. The desired monoclonal antibodies were obtained by using as immunogen a synthetic peptide (PF2) similar to the P1.2 C-terminus coupled to ovalbumin. The P1.2 C-terminus is a unique epitope not present on the parent protein of F1.2, prothrombin. The approach of obtaining site-directed antibodies by using as immunegen a synthetic peptide resembling a known primary sequence coupled to a carrier protein has been reviewed (19). Examples in which this approach was successful for generating monoclonal antibodies specific for an activation product but not its parent protein include antibodies reactive with fibrin but not fibrinogen (20) and antibodies reactive with trypsinogen activation peptide but not trypsinogen (21). In these examples and our anti-F1.2 antibodies, the neoepitope emulated by the synthetic peptide immunogen was a newly exposed terminus present on the activation product but not on the zymogen. CLINICAL
CHEMISTRY,
Vol. 39, No. 4, 1993
589
The
sequence
amino
acids
(SDRAIEGR) of our
for the
immunogen
peptide
eight
PF2
C-terminal
was based
on the
cDNA sequence of prothrombin (8) and the cleavage sites of prothrombin. For conjugation purposes, two additional amino acids (CG) were added to the PF2 N-terminus. Our synthetic peptide differs in both length and sequence from that which Pelzer et al. (2) used to obtain anti-F1.2 polyclonal antibodies. The sequence of their 15-amino-acid synthetic peptide (CLDEDSDEERAIEGR) was based on amino acid analysis (22) and included two glutamic acid residues not predicted by cDNA analysis (8). The additional residues in their peptide immunogen may not have been critical for producing polyclonal antibodies that had reasonable F1.2 specificity after imniunopurification. However, we note that the glutamic acid residues interrupt the sequences of six to eight amino acids that constitute the known
critical epitope of our highly nal antibody 5-3B.
specific
anti-Pl.2
monoclo-
We
the use of synthetic peptide immunogens recognize, most importantly, the native protein being emulated. To ensure that this requirement was met with our antibodies, our early hybridoma screening included tests for reactivity against both immunogen peptide PF2 and human P1.2. Qualitatively, each of the six selected monoclonal antibodies recognized native P1.2; quantitatively, P1.2 and synthetic peptide PF2.14 competed comparably for immobilized antibody 5-3B binding sites. Therefore, the careful selection of synthetic peptide immunogen plus appropriate screening protocols allowed for isolation of monoclonal antibodies applicable for use in an P1.2 imlnunoassay. Monoclonal antibody 5-3B recognized not only native F1.2 but also denatured, reduced proteins bearing the C-terminal sequence of SDRAIEGR, as demonstrated by Western blot reactivity. The ability of this antibody to recognize denatured F1.2 and F2 is not surprising, given that the antibody was raised against a synthetic peptide (PF2) immunogen representing a linear C-terminal epitope on P1.2. Epitope mapping of monoclonal antibody 5-3B showed that the presence of both a C-terminal arginine with a free carboxyl group and six to eight specific amino acids resembling the P1.2 C-terminus were critical for reactivity. Because of its identical C-terminal sequence to F1.2 (Table 1), F2 did bind to antibody 5-3B, as expected. On the other hand, it is unlikely that antibody 5-3B binds to either des-RP1.2 or prothrombin fragment 1.2.3 (P1.2.3) because of differences between their C-terminal sequences and that of F1.2. Des-RF1.2 is a proposed F1.2 degradation product missing the terminal arginine. Des-RF1.2 could theoretically exist in vivo because the F1.2 C-terminal GR sequence is a favorable site for carboxypeptidase activity (23). Fl.23 is a labile prothrombin activation peptide reportedly formed by cleavage at prothrombin R-T and has a C-terminal sequence distinctly different from F1.2 (24). The clinical significance, if any, of des-RF1.2 and P1.2.3 is unknown, Site-directed
590
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monoclonal
CHEMISTRY,
but neither would be measured in our 5-3B-based ELISA. In our ELISA, the high specificity of monoclonal antibody 5-3B allows quantification of 0.08 nniol/L P1.2 in the presence of 1.5 nnoIfL prothrombin, the amount typically found in plasma. HRP-labeled rabbit polyclonal antibodies to the P1.2 N-terminus serve as tagging antibodies in the assay. This particular combination of capture and label antibodies ensures specificity for P1.2 only. Polyclonal antibody-based assays previously described (2, 11) measure both FL2 and its potential degradation product F2. The similarity in the mean concentrations of plasma P1.2 in healthy individuals measured by our assay and these other, less-specific assays suggests that P2 may not be a major degradation product in healthy individuals. However, the extent to which plasma F2 may exist in unhealthy populations remains unclear.
antibodies
obtained must
Vol. 39, No. 4, 1993
from
have described the first monoclonal antibodyassay for plasma P1.2, a key biological marker of prothrombin activation and thrombin formation. The ELISA, performed on routinely collected heparinized plasma, is sensitive, specific, and reproducible and requires only commonly available micro-ELISA equipment. The assay should be a useful method for future clinical studies assessing the role of P1.2 in defining the prethrombotic state, assessing thrombotic risk, and monitoring the efficacy of anticoagulant therapy. based
We thank Patricia A. Byrd for her valuable technical contributions, Charles Greenberg for his critical review of this paper, and David Sane for providing certain thrombolytic agents.
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