ANALYTICAL BIOCHEMlSTRY 147, 529-534 (1985)

Enzyme-Linked Coagulation Assay: A Clot-Based,

Solid-Phase Assay for Thrombin’
GEORGE J. DOELLGAST*,~ AND HENRY ROTHBERGER~
With the technical assistance of Julie A. Luttinger

*Department of Biochemistry and Section on Rheumatology, fDepartment of Medicine, Bowman Gray School of
Medicine, Wake Forest University, Winston-Salem, North Carolina 27103
Received January 25, 1985

A new, solid-phase microtiter plate assay for thrombin has been developed, using fibrinogen
bound to wells of a microtiter plate and peroxidase-fibrinogen in solution as an indicator
system. When small amounts of thrombin are added to the mixture, peroxidase-fibrin and
plate-bound fibrin are formed, and the peroxidase-fibrin binds to the plate-bound fibrin. The
amount of peroxidase-fibrin binding is proportional to the thrombin concentration and time
of incubation. Using this assay,thrombin was measured at concentrations as low as 0.25 ng/
ml (0.006 nM) in 150 ~1 of sample. In the presence of the specific inhibitors benzamidine and
o-pbenylalanyl-L-prolyl-L-arginine chloromethyl ketone, the thrombin activity is reduced, at
relative concentrations of inhibitors consistent with their affinities and mechanisms of action.
The enzyme-linked coagulation assay is generally useful as a highly sensitive and convenient
alternative to conventional “clot-based’ tests of coagulation. 0 1985 Academic PWS. I~C.
KEY WORDS: thrombin: clotting assays:solid-phase assays.

Coagulation is a complex process, resulting tor of interest. Typically, human plasmas

from activation of a cascade of specific pro-
teases and effector proteins (1). Its end prod-
uct is the development of a clot due to
fibrinogen cleavage to fibrin by the specific
protease thrombin, followed by polymeriza-
tion of fibrin and formation of a stabilized,
crosslinked gel. Measurement of coagulation
most commonly uses the formation of a
fibrin clot as an endpoint indicating thrombin
activity (2), and these assays are therefore
called “clot-based.” Such assays can quanti-
tate any factor of the coagulation cascade in
a mixture of components of the relevant
pathway using substrates deficient in the fac-
’ Supported in part by Grants R01 CA 23533-06 and
ROI AM HL 21940-04 from the National Institutes of
Health, Bethesda, Maryland. U. S. Patent application is
pending on the solid-phase assay of coagulation factors.
‘To whom reprint requests should be addressed at
Department of Biochemistry, Bowman Gray School of
Medicine, Wake Forest University, 300 S. Hawthorne
Road, Winston-Salem, N. C. 27103.
genetically deficient in a single factor or
mixtures of factors purified from plasma are
the substrates employed for these clot-based
tests (3). A major alternative to clot-based
techniques is the use of chromogenic, syn-
thetic peptide substrates. These peptides are
structural analogs of the natural substrates of
the activated clotting factors of interest (4).
We wished to develop an alternative clot-
based test which would be more sensitive
and easy to perform on multiple samples. In
considering how to accomplish this objective,
we noted that fibrinogen binds extremely
tightly to plastic surfaces (5-7), and further
that, when bound to plastic surfaces and
converted to fibrin, solid-phase fibrinogen
can bind fibrin from solution (8,9). The
simultaneous conversion of solid-phase and
solution-phase fibrinogen to fibrin thus results
in the binding of some of the solution-phase
fibrin to the solid phase. Solid-phase fibrin
has been used as a substrate for assay of

529 0003-2697185 $3.00

Copyright 0 1985 by Academic Press. Inc.
All rights of repmduction in any form reserved.
530 DOELLGAST AND ROTHBERGER

plasmin and other proteases (10). If plastic
coated with fibrinogen were mixed with la-
beled fibrinogen in solution, and they were
both activated to fibrin by added thrombin,
then the initial polymerization reaction
following fibrinogen-fibrin conversion by
thrombin would be measurable as binding of
labeled fibrin to the microtiter plate. This
solid-phase association is strictly analogous
to polymerization in solution phase, and
should therefore be equivalently applicable
for clot-based tests.
Our choice of label was based on the
widely accepted technique of enzyme-linked
immunosorbent assay (ELISA),3 which uses
enzyme-labeled antigens and antibodies for
measurement of immunochemical reactions
( 1 1- 13). The advantages of this technique of
labeling include low cost, stability of reagents,
and ease of measurement using photometers
( 14), especially the microtiter plate readers
produced by several manufacturers. In this
report, we describe and validate the devel-
opment of an enzyme-linked coagulation as-
say (ELCA) for thrombin, using peroxidase-
labeled fibrinogen and fibrinogen coated on
microtiter plate wells as a substrate for
thrombin.
MATERIALS AND METHODS
Reagents. Human thrombin (Cat. No. T-
6759), horseradish peroxidase (Type VI, Cat.
No. P-8375), and bovine serum albumin
(Fraction V, Cat. No. A-9647) were obtained
from Sigma Chemical Company, St. Louis,
Missouri. DPhenylalanyl-L-prolyl-L-arginine
chloromethyl ketone (PPACK) was purchased
from Calbiochem-Behring, San Diego, Cali-
fornia. All other chemicals were the highest
grade commercially available.
3 Abbreviations used: ELISA, enzyme-linked immu-
nosorbent assay;ELCA, enzyme-linked coagulation assay; at 37°C for 60 min. The peroxidase-aldehyde
PPACK, D-phenylalanyl-L-prolyl+arginine chloromethyl was separated on a 1.5 X 30-cm column of
ketone; TABS, Tris-acetate-buffered saline: BSA, bovine
serum albumin; OPD, o-phenylenediamine: OD, optical
density; Th- I, amount of thrombin yielding an OD value dium
of 1 in the ELCA assay under a given set of conditions. fibrinogen and incubated at 37°C for 2 h.
Preparation of jibrinogen. Two liters of
titrated plasmapheresis plasma were precip-
itated with 100 ml of 1 M barium chloride
containing 10 mM benzamidine to remove
vitamin K-dependent factors (15). This sus-
pension was mixed on a roller apparatus at
4°C for 2 h and then centrifuged at 15OOg,,,
for 60 min. The supernatant was passed
through a 4.3 X 40-cm column of gelatin-
Sepharose to remove fibronectin (16), and
then through a 4.3 X 40-cm column of lysine-
Sepharose to remove plasminogen (17). The
supernatant was precipitated with 10% PEG-
1000 ( 18), centrifuged at 15OOg,,, for 60
min, and dissolved in 200 ml of 10 mM
EDTA, 10 mM benzamidine, 10 mM Tris-
HCI, saline, pH 7.6. The fibrinogen was
redissolved and precipitated twice more with
10% PEG- 1000 and passed through a 2.5
X 115-cm column of agarose A- 1.5m equili-
brated with the EDTA-benzamidine-buffered
saline. The purified fibrinogen was then stored
at -70°C in the EDTA-benzamidine-buff-
ered saline containing 30% glycerol. Fibrin-
ogen concentration was estimated from the
absorbance at 280 nm [extinction coefficient
of a 1% solution taken as 15.5 (19)]. Fibrin-
ogen was 95-98% clottable and was electro-
phoretically pure, as reported (18).
Labeling of fibrinogen with peroxidase. A
modification of the peroxidase labeling tech-
nique described by Nakane and Kawaoi (20)
was used for the labeling of human fibrinogen.
A 300-mg amount of fibrinogen (15 mg/ml)
was dialyzed against 1 liter of 0.05 M sodium
bicarbonate, pH 8.5, for 4 h at 4°C. Then
0.2 ml of 1% dinitrofluorobenzene was added
to 40 mg of horseradish peroxidase in 2 ml
of 0.05 M sodium bicarbonate, and the mix-
ture was incubated at 37°C for 30 min. A
0.2-ml amount of 0.5 M sodium metaperiod-
ate was added, and the mixture was incubated
Sephadex G-25 equilibrated with 0.05 M so-
bicarbonate and was added to the
 SOLID-PHASE THROMBIN ASSAY 531

The Schiff base was reduced by addition of using 50 ~1 of 1 M H2S04, and absorbance
50 mM sodium borohydride to a final con- was measured using a microtiter plate reader
centration of 10 mM, and the conjugate was (Bio-Tek) at 490 nm.
dialyzed overnight against EDTA-benzami-
dine-buffered saline and separated on a 2.5 RESULTS
X 115-cm column of agarose A-l .5m equil-
The activity of thrombin in this assay is
ibrated with the EDTA-benzamidine-buffered measured by the production of peroxidase-
saline. The molar ratio of peroxidase to fibrin and its attachment to the plate. Figure
fibrinogen was 0.39. This preparation was
1 shows the results for five different incuba-
diluted 3.3-fold with fibrinogen and stored tion times; three stages are apparent. Below
as a stock solution containing 0.72 mg/ml a critical threshold of thrombin concentration
fibrinogen with a molar ratio of 0.12 mol of and length of incubation, no peroxidase-
peroxidase per mole of fibrinogen. This was
fibrin binds to the plate. When the threshold
the 100X stock solution used in all subsequent
thrombin concentration is reached, some
experiments. peroxidase-fibrin binds, but the amounts
Measurement of thrombin using ELCA.
bound are sufficiently small that optical den-
Plates were coated with 150 ~1 per well of 50
sity (OD) values less than 2 are obtained in
pg/ml of fibrinogen in 10 mrvr Tris-acetate-
the peroxidase assay. Finally, relatively large
buffered saline (TABS) containing 10 mM amounts of peroxidase-fibrin are deposited
EDTA, pH 7.6, overnight at 4°C. Then the
on the plate, and OD values are off-scale (>2).
solution was removed from the plate. Incu-
This formation and deposition of peroxi-
bations were performed for the indicated
dase-fibrin mediated by thrombin are anal-
times in the TABS buffer without added
ogous to the coagulant activity of thrombin.
EDTA at 37°C. To ensure rapid temperature
Only one incubation time is used for each
equilibration, plates were floated in a water
plate, and each sample is serially diluted until
bath. Bovine serum albumin (BSA) was in-
the amount of peroxidase-fibrin detected on
cubated at pH 1.5 for 20 min at 37°C and
then brought to pH 7.6 at a concentration
of 100 mg/ml. This stock solution of BSA
was added to the ELCA incubation mixture
2r
at a final concentration of 2.5 mg/ml. Per-
1.6
oxidase-labeled fibrinogen was added at a
1: 100 dilution of the stock solution. Throm- 1
bin was added to the assay mixture, and g1.2-
z
incubation was for lo-160 min at 37°C.
After incubation, the reaction mixture was d .8-
shaken from the microtiter plate wells, and d
the plate was rinsed with saline. When assays
.4-
were performed comparing thrombin activity
in the presence of inhibitors, all samples were
assayed in the same plate.
Assay of peroxidase used 0.15 ml of a
substrate concentration of 0.0 15% hydrogen
FIG. 1. Assay of thrombin at varying incubation times
peroxide in 0.3 M Tris-citrate, pH 6.0, con- using ELCA. Peroxidase-fibrinogen and BSA were pi-
taining 0.5% Triton X-100, using 0.6% o- petted onto the plate, and thrombin was added and
phenylenediamine (OPD) as the indicator incubated at 37°C. Incubation times are indicated on
dye. The reaction was terminated after 8 min the figure.
532 DOELLGAST AND ROTHBERGER

the plate is below maximal levels. A conve- % Inhibition = 100 X (1 - ro/Ti)

nient representation of the concentration of
where To is the Th-1 value in the absence of
thrombin at which this occurs is the amount
inhibitor, and Ti is the Th-1 value at a given
of added thrombin yielding an OD value of
inhibitor concentration.
1, defined as the Th-I value. This is seen in
Figure 3 presents the inhibition of throm-
Table 1; this value varies from 0.02 to 0.0008
NIH units/ml (6.3-0.25 rig/ml) for incubation bin by benzamidine and PPACK, calculated
in this way. The inhibition by benzamidine
times from 20 to 160 min.
reaches a level of 50% at a concentration of
Variability of the assay for thrombin was
1.8 mM, which is an order of magnitude
examined. Comparison of 12 replicate sam-
greater than the Ki value determined using
ples of thrombin assayed for 40 min on three
synthetic substrates in an esterolytic assay
plates yielded within-plate coefficients of
(21). This is consistent with the results of
variation of 6.1, 5.0, and 7.8%. Between-
Markwardt et al., who found (22) that the
plate variation of the mean value on the
concentrations of benzamidine required to
three plates had a coefficient of variation
inhibit the coagulant activity of thrombin by
of 7.6%.
50% was 20-fold higher than its Ki defined
Two widely used inhibitors of thrombin
in an esterolytic assay. The concentration of
are benzamidine, a reversible competitive
PPACK which inhibits thrombin by 50% (30
inhibitor (2,2 1,22), and PPACK, an irrevers-
PM) is equivalent to the thrombin concentra-
ible, active site inhibitor (23). The effects of
tion (27 PM), and is nearly 8 orders of
these inhibitors added in increasing concen-
magnitude lower than the concentration of
trations to an 80-min thrombin assay are
benzamidine which is 50% inhibitory. Equiv-
seen in Fig. 2. At increasing concentrations
alent results were obtained for incubation
of inhibitor, the Th-1 value increases. If the
times of 40 and 120 min, except that the
Th-I value doubles, then twice as much
50% inhibitory value for benzamidine did
thrombin is needed to obtain equivalent ac-
not vary, but the corresponding value for
tivity, and thrombin is 50% inhibited; if
PPACK was proportional to the Th-1 value,
increased by 10X, then inhibition is 90%.
i.e., the molar ratio of PPACK to thrombin.
The inhibition of thrombin can thus be cal-
culated from this value as
DISCUSSION

TABLE I In this work, we have described enzyme-

linked coagulation assay, a sensitive, specific
ASSAY OF THROMBIN USING ELCA. AT VARYING assay for thrombin. Since ELCA uses fibrin-
CONCENTRATIONS OF THROMBIN AND
TIME OF INCUBATION ogen as a substrate and fibrin polymerization
as an endpoint, it is comparable to other
Incubation Thrombin concentration clot-based tests using purified fibrinogen or
time (min) yielding an OD490 = 1 plasma as substrate and clot formation as the
measured endpoint of the assay.
10 >o. I
20 0.02
Advantages of the ELCA include high
40 0.005 sensitivity (less than 1 rig/ml of thrombin
80 0.002 can be measured) and convenience (multiple
160 0.0008 samples can be assayed simultaneously, using
microtiter plate readers to measure the re-
Note. Data are from Fig. 1, showing the concentrations
of thrombin resulting in an OD,,, of 1.0 under these sults). Reproducibility is good, as seen by
assay conditions. Thrombin activity is expressed as NIH examination of within-plate and between-
units/ml. plate variability.
 SOLID-PHASE THROMBIN ASSAY 533

6 .8 6 .8-
d d

.4-

A Thrombin INIH units/ml)

FIG. 2. Assay of thrombin using ELCA in the presence of varying concentrations of inhibitors. (A)
Assays in the presence of benzamidine; (B) assaysin the presence of PPACK. Incubation was for 80 min
at 37°C. Concentrations of inhibitors, in mM (benzamidine) or nM (PPACK). are indicated on the figure.
“Cont 1” is the control in the absence of inhibitor.

ELCA requires long incubations (>20 min)
to optimize the solid-phase association of
peroxidase-fibrin, and all assays are per-
formed by using multiple dilutions of sam-
ples. Classical clotting tests are standardized
using multiple dilutions of standard and
measuring the time required to form a clot
(usually less than 1 min); unknown samples
are compared with the standard clotting time.
ELCA is equivalent to testing multiple sam-
lE-08 lE-I36 .OOOl .Dl
IInhIbitor
FIG. 3. Inhibition of thrombin in an ELCA assay by
benzamidine and PPACK. Data of Fig. 2 were used to
calculate percentage inhibition, as indicated in the text.
ples by performing a dilution series for each
sample and finding the concentration of sam-
ple yielding a clotting time identical to that
of the standard.
The solid-phase assay of thrombin could
in principle employ any labeling technique
for derivatization of fibrinogen, coupled with
a suitable detection system. In the ELCA
method, enzyme labeling of fibrinogen has
the same advantages that attend the use of
enzyme-labeled antigens and antibodies in
ELISA techniques, i.e., stability of reagents
and low cost of photometers for detection.
Fluorescent labels could also be used very
conveniently with fluorometric plate readers,
and of course radiolabeled fibrinogen could
be substituted, with somewhat less conve-
nience but with at least equal precision.
The use of ELCA for assay of factors other
than thrombin could readily be accomplished
by a two-stage assay, utilizing ELCA to mea-
sure the thrombin generated, or as a one-
1 stage assay with plasmas or purified factors
ImMl present in the microtiter well. Both ap-
proaches could take advantage of the high
sensitivity and specificity of thrombin assay
using ELCA. In our own studies, we have
534 DOELLGAST AND ROTHBERGER
found the latter approach to be a convenient
and sensitive alternative to one-stage clotting
tests (Doellgast and Rothberger, in prepara-
tion).
ACKNOWLEDGMENT
We appreciate the helpful comments and critical review
of this manuscript by Dr. Roy Hantgan of the Department
of Biochemistry.
REFERENCES
1. Davie, E. W. (1981) in Methods in Enzymology
(Lorand, L., ed.), Vol. 80, pp. 153-156, Academic
Press, New York.
2. Workman, E. F., and Lundblad, R. L. (1980) in
CRC Handbook Series in Clinical Laboratory
Science, Section I, Hematology (Seligson, D.. ed.;
Schmidt, R. M., section ed.), Vol. III, pp. I49-
169, CRC Press, Boca Raton, Fla.
3. Rothberger, H., McGee, M., and Lee, L. T-K. (1984)
J. Clin. Invest. 73, 1524-1531.
4. Fareed, J.. Messmore, H. L., Waleng, J. M., and
Bermes, E. W. (1983) Clin. Chem. 29, 225-236.
5. Parsons, G. H. (198 I) in Methods in Enzymology
(Langone, J. J., and Van Vunakis, H., eds.), Vol.
73, pp. 224-239, Academic Press, New York.
6. Pesce, A. J., Ford, D. J., Gaizutis, M., and Polak,
V. E. (1977) Biochim. Biophys. Acta 492, 399-
407.
7. Morrisey, B. W. (1977) Ann. N. Y. Acad. Sci. 283,
50-64.
8. Ihlenfeld, J. V., and Cooper, S. L. (1979) J. Biomed.
Mater. Rex 13, 577-591.
9. Packman, M. A., Evans, G., Glynn, M. F., and
Mustard, J. F. (1969) J. Lab. Clin. Med. 73,686-
697.
10. Moroz, L. A., and Gilmore, N. J. (1975) Blood 46,
543-555.
1 I. Engvall, E., and Perlmann, P. (197 1) Immunochem-
istry 8, 87 l-874.
12. Engvall, E., Jonsson, K., and Perlmann, P. (1971)
B&him. Biophys. Acta 251, 427-434.
13. Engvall, E., and Perlmann, P. (1972) J. Immunol.
109, 129-135.
14. Engvall, E. (1980) in Methods in Enzymology (Van
Vunakis, H., and Langone, J. J., eds.), Vol. 70,
pp. 419-439, Academic Press, New York.
15. Broze, G. J., and Majerus, P. W. (1980) J. Biol.
Chem. 255, 1242- 1247.
16. Engvall, E., and Ruoslahti, E. (1977) Int. J. Cancer
20, l-5.
17. Deutsch, D. G., and Mertz, E. T. (1970) Science
(Washington, D. C.) 170, 1095-1096.
18. Maxi, M. A., Masri, S. A., and Boyd, N. D. ( 1983)
Thromb. Haemostasis 49, 116-I 19.
19. Doolittle. R. F. (1980) in CRC Handbook Series in
Clinical Laboratory Science, Section I, Hematology
(Seligson, D., ed.; Schmidt, R. M., section ed.),
Vol. III, pp. 3-14, CRC Press, Boca Raton, Fla.
20. Nakane, P. K., and Kawaoi, A. (1974) J. Histochem.
Cytochem. 22, 1084-1091.
2 I. Geratz, J. D., and Tidwell, R. R. (1977) in Chemistry
and Biology of Thrombin (Lundblad, R. L., Fen-
ton, J. W., and Mann, K. G., eds.), pp. 179-196,
Ann Arbor Science Publishers, Ann Arbor, Mich.
22. Markwardt, F., Landmann, H., and Walsmann, P.
(1968) Eur. J. Biochem. 6, 502-506.
23. Kettner, C., and Shaw, E. (1979) Thromb. Res. 14,
969-973.