HYBRIDOMA Volume 10, Number 5, 1991 Mary Ann Lieben. Inc., Publishers
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
Monoclonal Antibodies Against Human Antithrombin III HANA CAJTHAMLOVÁ, Jlftí T. NOVÁK, EDUARD PALUSKA, and PETR STÖCKBAUER
ZBYNËK HRKAL, Institute
of Hematology and Transfusion,
U nemocnice 1, CS 128 20 Praha 2, Czechoslovakia
ABSTRACT Three monoclonal antibodies identified as D8, B11 and C5 of different specificities have been produced against human antithrombin III The apparent dissociation constants (K app) of the AT antibody interaction were determined by ELISA method: K, app (D8)= = 2.h nmole, K app 13 nmole, K app (C5) = 2k nmole. All three antibodies reacted with isolated AT on immunoblots obtained with "native" PAGE. The D8 antibody also reacted with plasma and serum AT while B11 antibody reacted with serum thrombin-antithrombin complexes as well.
(AT).
-
(B11)
(TAT)
INTRODUCTION Antithrombin III (AT) is considered to be the main physiological inhibitor of the coagulation proteinases of the serine type. By formation of the covalently linked complexes, this plasma protein in cooperation with heparin inhibits thrombin and factor Xa (1-3) as well as other activated coagulation proteinases like factors IXa, XIa, Xlla and also plasmin and kallikrein (k-6). In this way it regulates the coagulation processes limiting them to proceed in the vicinity of the site of tissue damage. Under pathological conditions (liver damage, enteropathy, nephropathy) or if the coagulation process is deregulated (hypercoagulation states, disseminated intravascular coagulation) or the functionally defective AT molecules are synthesized (mutant AT's) the concentration and/or AT activity may be lowered (7-10). While the AT activity is commonly measured through the residual amidolytic activity of thrombin added in definite amount to the examined plasma (11,12), the total AT level can directly be determined by immunochemical methods of which EIA employing polyclonal anti AT antibodies is mostly used (13»1*0« Recently Knoller and Savion (15) produced and characterized four monoclonal antibodies directed towards different epitopes of AT.
For the development of ELISA method of AT determination as as for searching for abnormal antithrombins by PAGE and immunoblotting, the monoclonal antibodies appear to be reagents of choice. We developed and characterized three monoclonal anti AT III antibodies which might be suitable for these purposes.
well
633
MATERIALS AND METHODS Materials
Heparin Sepharose CL-6B, Protein A-Sepharose CL-6B and SDS were products of Pharmacia-UCB, Sweden, Mouse Typer Isotyping Kit, nitrocellulose membrane (0.45 um), SDS, acrylamide and bisacrylamJ.de were products of Bio-Rad, USA, HAT medium was purchased from Sigma, USA and peroxidase labeled swine anti mouse IgG was obtained from the Institute of Sera and Vaccines, molecular markers
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
Czechoslovakia.
Isolation of antithrombin III AT was isolated from fresh
or frozen human plasma by chromatography on heparin Sepharose CL-6B (16) followed by chromatography of the fraction with AT activity on Superose-Q in Tris-HCl buffer pH 8.0 with linear gradient to 0.4 M NaCl in the same buf-
fer. Production of monoclonal antibodies White Balb/c mice wereimmunized by three consecutive injections with 100 ug of highly purified AT at two-weeks intervals. Three to five days after the last immunization dose in complete Freund s adjuvant, mice were splenectomized and the lymphocyte suspension (5x107 cells) was mixed with 2x10 cells of the SP-2/0-Ag 1k myeloma cell line. The fusion was performed according to Köhler and Milstein (17)- The cell suspension was cultivated in HAT medium and the individual colonies were screened for antibodies production against AT by means of ELISA technique. The recloned hybridomas were inoculated into perithoneum of the Balb/c mice and in 7 to 10 days the ascites fluids were collected.
Isolation of the monoclonal antibodies The ascites fluids were centrifugea at 100 000 x g for 30 min and dialyzed to 20 mM Tris-HCl buffer pH 7«2. Isolation of immunoglobulins was performed on Protein A Sepharose by consecutive elution of the bound proteins with 0.1 M citric acid adjusted to pH 6, 5i and k as described by Fredricksson et al. (18). The anti AT III IgG's were dialysed to TBS pH 8.0, concentrated by ultrafiltration to 1.0 mg/ml solution and stored frozen at -50 C in 0.5 nil
aliquots. Determination of IgG subclasses The immunoglobulin subclasses were determined nique employing the Mouse Typer Isotyping Panel of
by ELISA techBio-Rad, USA.
Electrophoresis and immunoblotting The polyacrylamide gel electrophoresis (PAGE) was performed using Mini-Protean II (Bio-Rad, USA) gel electrophoresis system in 7«5% "native" gels and 12% SDS gels in discontinous buffer system
(19).
To the wells
10 ul of 1
mg/ml
AT solution
or
1
:
1
diluted
plasma or serum was applied. The gels were stained with Coomassie blue R-25O or they were blotted onto nitrocellulose membrane in 48 mM Tris, 39 mM Glycine, 1.3 mM SDS, 20% Methanol pH 9.2 transfer buffer (20) employing semi-dry electrotransfer apparatus, Multiphor II NovaBlot system by Pharmacia-LKB, Sweden. The blots were
reacted with the individual monoclonal antibodies (the 1 mg/ml stock solutions of IgG's diluted 1/100 to I/500) and developed with peroxidase labeled swine anti mouse IgG employing 4-chloro-1 naphtol as the chromogenic substrate. Crossed irnmunoelectrophoresis in agarose gels containing polyclonal rabbit anti AT antibodies with and without heparin incorporated into the first dimension gel was performed in 50 mM Trisbarbital pH 8.8 buffer.
634
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
Determination of AT III activity/concentration The AT activity was determined on the basis of its ability to inhibit amidolytic activity of thrombin added in definite amount to the system. As the chromogenic substrate, Z-Gly-Pro-Arg-pNA was used (21). Concentration of AT was determined by the EIA method using rabbit polyclonal anti AT antibodies (22). Antithrombin III determination by ELISA Dynatech 96 well microtiter plates were coated with 100 ul of the 11 or 22 nM/ml AT solution. After overnight incubation and three washings, the unoccupied binding sites were blocked with 0.02% gelatin solution for 60 min. After three washings with PBS, 0.05% Tween 20, 100 ul of the first anti AT monoclonal antibody (concentration 0.1 ng/ml to 10 ug/ml) was added and incubated for 90 min at 37°C. After three washings with PBS-Tween 20 solution, 100 ul of the second anti AT monoclonal antibody at a fixed concentration of 2 ug/ml was added and incubated for 90 min at k C. Finally, the swine anti mouse peroxidase labeled IgG was added and after 90 min incubation, the visualization with OPD H„0„ system was performed. The binding curves were computer fitted by nonlinear regression using Hill s equation employing GraphPAD, ISI, USA, computer program. -
RESULTS AND DISCUSSION Three monoclonal antibodies named D8, C5 and B11 have been constructed and isolated and the binding profiles against isolated AT were determined by ELISA technique. Fig. 1 A shows the binding curve of D8 monoclonal antibody while 1 B shows the same experiment performed after the AT coated plate had been first incubated with the C5 monoclonal at a concentration exceeding the saturation level and the excess of the first antibody was washed out. Similarly the binding curves for the interaction of AT with C5 monoclonal antibody is given in Fig. 2 A and with C5 after saturation with D8 in Fig. 2 B. In Table 1 the binding parameter values are given for the binding of monoclonal antibodies to antithrombin III. TABLE 1 The
apparent
(K, app)
dissociation constants of the AT-antibody complexes
Monoclonal
Subclass
antibody
K,
app
(nmolej
D8
IgG
2.4
B11
IgG1 IgG1
13.0 24.0
C5
It follows from the above experiments that D8 and C5 antibodies bind independently and, therefore, that they are directed to the different epitopes on AT molecule. The binding properties of the antibodies were further examined by "native" and SDS PAGE of isolated antithrombin, of human plasma and of human serum by immunoblotting technique. Fig. 3 shows the immunoblots of the PAGE
635
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
1.0
o
en
0.5
0.0 -1
2
1
log
c
(D8) tog/1]
o
01
A. Binding profile of antithrombin III D8 monoclonal antibody interaction obtained with ELISA method. D8 monoclonal interaction B. Binding profile of AT after AT saturation with C5 antibody.
FIGURE 1
-
-
636
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
o
O)
3
2
log
c
(C5) [//g/1]
o O)
3
2
log
c
(C5) [//g/1]
FIGURE 2 A. Binding profile of the antithrombin III C5 monoclonal antibody interaction. B. Binding profile of the AT C5 interaction after AT saturation with D8 monoclonal antibody. -
-
637
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
(p)
and isolated FIGURE 3« Immunoblots of human serum (S), plasma antithrombin III following "native" PAGE developed with D8 and B11 monoclonal antibodies.
(AT)
of isolated AT, serum and plasma developed with D8 and B11 monoclonal antibodies. It follows that while the D8 antibody reacts with the isolated AT as well as with AT of plasma and serum, the B11 antibody reacts predominantly with TAT complexes, so the B1 1 antibody must be directed towards an epitope different than the D8 antibody. The reactivity of the antibodies judged from electrophoretic experiments is summarized in Table 2. TABLE 2
Reactivity
of the anti AT monoclonal antibodies with AT after PAGE and electroblotting
antibody
PAGE "native" serum AT plasma AT TAT AT
D8
X
X
X
B11
X
X
X
C5
X
Monoclonal
SDS PAGE AT
AT(r) X
In conclusion: three different monoclonal antibodies against AT were constructed and isolated of which D8 is apparently the most useful. The detection limit on immunoblots with peroxidase detection is below 1 ug. It is directed towards the sequential epitope present both on native as well as on SDS denatured and reduced molecule which is not influenced by heparin binding. The C5 monoclonal reacts with the isolated AT about 10 times less strongly and no reactivity was observed towards plasma AT on immunoblots. This antibody is apparently directed to the conformational epitope since no reactivity was observed towards SDS denatured AT on immunoblots. The B11 antibody has lower avidity for AT than D8 antibody, it reacts, however, not only with AT in antithrombin complexes plasma and serum but also with thrombin -
638
(TAT). So it appears that the epitope of the AT molecule towards which the B11 antibody is directed is not influenced by the antithrombin-thrombin interaction. A CKNOWLE DGMENTS
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
Ms
The authors wish to thank Ms Sedlmaierová, Ms Velebná and Benesová for their skillful technical assistance. REFERENCES
1. 2.
3.
4. 5.
6. 7.
8.
9.. 10. 11.
ABILDGAARD, U. (1968). Highly purified antithrombin III with heparin cofactor activity prepared by disc electrophoresis. ROSENBERG, R.D., and DAMUS, P.S. (1973)- The purification and mechanism of action of human antithrombin-heparin cofactor. J. Biol. Chem. 248, 6490. OWEN, W.G., PENICK, G.D., YEDER, E., and P00LE, B.L. Evidence for an ester bond between thrombin and heparin cofactor. Thromb. Haemostas. 35» 87. ROSENBERG, J.S., McKENNA, P., and ROSENBERG, R.D. (1975). Inhibition of human factor IXa by human antithrombin-heparin cofactor. J. Biol. Chem. 250, 8883. STEAD, N., KAPLAN, A.P., and ROSENBERG, R.D. (1976). Inhibition of activated factor XII by antithrombin-heparin cofactor. J. Biol. Chem. 251, 6481. VENNEROD, A.M., LAAKE, K., SOLBERG, A.K. and STROMLAND, S. (1975)- Inactivation and binding of human plasma kallikrein by antithrombin III and heparin. Thromb. Res. 9, 457« BULLER, H.R., and CATE, J.W. (1989). Acquired antithrombin III deficiency: Laboratory diagnosis, incidence, clinical implications and treatment with antithrombin III concentrate. Am. J. Med. 87 (suppl 3B), 44S. BERESFORD, C.H. (1988). Antithrombin III deficiency. Blood reviews 2, 2 39. PROCHOWNIK, E. (1989). Molecular genetics of inherited antithrombin III deficiencies. Am. J. Med. 87 (suppl 3B)> 15S. HIRSH, J., PIOVELLA, F., and PINT, M. (1989). Congenital antithrombin III deficiency. Am. J. Med. 87 (suppl 3B), 34s. ABILDGAARD, U., LIE, M., and ODEGABD, O.R. (1977). Antithrombin (heparin cofactor) assay with "new" chromogenic substrates (S-2238 and Chromozym- TH) Thromb. Res". 11, 539. HANDELAND, G.F., ABILDGAARD, U., and AASEN, A.O. (1983). Simplified assay for antithrombin III activity using chromogenic peptide substrate. Scand. J. Haematol. 31» 427. SAS, G., PEPPER, D.S., and CASH, J.D.(1975). Investigations on Antithrombin III in normal plasma and serum. Br. J. Haematol.
(19?6).
.
12.
13.
30, 265.
E.J. (198O). Immunological analysis of active and inactive antithrombin III. Br. J. Haematol. 46, 2 77. 15. KNOLLER, S., and SAVION, N. (1989)• Monoclonal antibodies against antithrombin III. Eur. J. Biochem. 180, 319« 16. McKAY, E.J. (1981). A simple two step procedure for the isolation of antithrombin III from biological fluids. Thromb. Res. 21, 37517. KÖHLER, G., and MILSTEIN, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495.
14. McKAY,
18.
FREDRIKSSON, U.B., FAGERSTAM, L.G., COLE, A.W.G., andLUNDGREN, (1986). Protein A-Sepharose CL-4B affinity purification of
T.
IgG monoclonal antibodies from mouse ascites. 6th International Congress of Immunology, Toronto (Poster). 19. LAEMMLI, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. 20. BJERUM, 0,J., and SCHAFER-NIELSEN, C. (1986). Buffer systems
639
and transfer parameters for semidry electroblotting with a horizontal apparatus. In: Electrophoresis 86. Dunn M.J. (ed).
Verlag Chemie, Weinheim, p. 31 5 BARTL, K., and LILL, H. (1980). A versatile assay method of antithrombin III using thrombin and Tos-Gly-Pro-Arg-pNA. Thrombosis Res. 18, 267. BEDNARÍK, T., RYBAK, M. , L0STICKÍ", C, and DYR, J.E.(l987). •
21.
22.
Immunochemical determination of antithrombin III.
36,
Ceskoslov.
317.
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
Farm.
Address
to: Hrkal
reprint requests Dr.
Zbynék
Institute of Hematology and Blood Transfusion U nemocnice 1 Praha 2, CS 128 2 0 Czechoslovakia
640
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
Monoclonal Antibodies Against Human Antithrombin III HANA CAJTHAMLOVÁ, Jlftí T. NOVÁK, EDUARD PALUSKA, and PETR STÖCKBAUER
ZBYNËK HRKAL, Institute
of Hematology and Transfusion,
U nemocnice 1, CS 128 20 Praha 2, Czechoslovakia
ABSTRACT Three monoclonal antibodies identified as D8, B11 and C5 of different specificities have been produced against human antithrombin III The apparent dissociation constants (K app) of the AT antibody interaction were determined by ELISA method: K, app (D8)= = 2.h nmole, K app 13 nmole, K app (C5) = 2k nmole. All three antibodies reacted with isolated AT on immunoblots obtained with "native" PAGE. The D8 antibody also reacted with plasma and serum AT while B11 antibody reacted with serum thrombin-antithrombin complexes as well.
(AT).
-
(B11)
(TAT)
INTRODUCTION Antithrombin III (AT) is considered to be the main physiological inhibitor of the coagulation proteinases of the serine type. By formation of the covalently linked complexes, this plasma protein in cooperation with heparin inhibits thrombin and factor Xa (1-3) as well as other activated coagulation proteinases like factors IXa, XIa, Xlla and also plasmin and kallikrein (k-6). In this way it regulates the coagulation processes limiting them to proceed in the vicinity of the site of tissue damage. Under pathological conditions (liver damage, enteropathy, nephropathy) or if the coagulation process is deregulated (hypercoagulation states, disseminated intravascular coagulation) or the functionally defective AT molecules are synthesized (mutant AT's) the concentration and/or AT activity may be lowered (7-10). While the AT activity is commonly measured through the residual amidolytic activity of thrombin added in definite amount to the examined plasma (11,12), the total AT level can directly be determined by immunochemical methods of which EIA employing polyclonal anti AT antibodies is mostly used (13»1*0« Recently Knoller and Savion (15) produced and characterized four monoclonal antibodies directed towards different epitopes of AT.
For the development of ELISA method of AT determination as as for searching for abnormal antithrombins by PAGE and immunoblotting, the monoclonal antibodies appear to be reagents of choice. We developed and characterized three monoclonal anti AT III antibodies which might be suitable for these purposes.
well
633
MATERIALS AND METHODS Materials
Heparin Sepharose CL-6B, Protein A-Sepharose CL-6B and SDS were products of Pharmacia-UCB, Sweden, Mouse Typer Isotyping Kit, nitrocellulose membrane (0.45 um), SDS, acrylamide and bisacrylamJ.de were products of Bio-Rad, USA, HAT medium was purchased from Sigma, USA and peroxidase labeled swine anti mouse IgG was obtained from the Institute of Sera and Vaccines, molecular markers
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
Czechoslovakia.
Isolation of antithrombin III AT was isolated from fresh
or frozen human plasma by chromatography on heparin Sepharose CL-6B (16) followed by chromatography of the fraction with AT activity on Superose-Q in Tris-HCl buffer pH 8.0 with linear gradient to 0.4 M NaCl in the same buf-
fer. Production of monoclonal antibodies White Balb/c mice wereimmunized by three consecutive injections with 100 ug of highly purified AT at two-weeks intervals. Three to five days after the last immunization dose in complete Freund s adjuvant, mice were splenectomized and the lymphocyte suspension (5x107 cells) was mixed with 2x10 cells of the SP-2/0-Ag 1k myeloma cell line. The fusion was performed according to Köhler and Milstein (17)- The cell suspension was cultivated in HAT medium and the individual colonies were screened for antibodies production against AT by means of ELISA technique. The recloned hybridomas were inoculated into perithoneum of the Balb/c mice and in 7 to 10 days the ascites fluids were collected.
Isolation of the monoclonal antibodies The ascites fluids were centrifugea at 100 000 x g for 30 min and dialyzed to 20 mM Tris-HCl buffer pH 7«2. Isolation of immunoglobulins was performed on Protein A Sepharose by consecutive elution of the bound proteins with 0.1 M citric acid adjusted to pH 6, 5i and k as described by Fredricksson et al. (18). The anti AT III IgG's were dialysed to TBS pH 8.0, concentrated by ultrafiltration to 1.0 mg/ml solution and stored frozen at -50 C in 0.5 nil
aliquots. Determination of IgG subclasses The immunoglobulin subclasses were determined nique employing the Mouse Typer Isotyping Panel of
by ELISA techBio-Rad, USA.
Electrophoresis and immunoblotting The polyacrylamide gel electrophoresis (PAGE) was performed using Mini-Protean II (Bio-Rad, USA) gel electrophoresis system in 7«5% "native" gels and 12% SDS gels in discontinous buffer system
(19).
To the wells
10 ul of 1
mg/ml
AT solution
or
1
:
1
diluted
plasma or serum was applied. The gels were stained with Coomassie blue R-25O or they were blotted onto nitrocellulose membrane in 48 mM Tris, 39 mM Glycine, 1.3 mM SDS, 20% Methanol pH 9.2 transfer buffer (20) employing semi-dry electrotransfer apparatus, Multiphor II NovaBlot system by Pharmacia-LKB, Sweden. The blots were
reacted with the individual monoclonal antibodies (the 1 mg/ml stock solutions of IgG's diluted 1/100 to I/500) and developed with peroxidase labeled swine anti mouse IgG employing 4-chloro-1 naphtol as the chromogenic substrate. Crossed irnmunoelectrophoresis in agarose gels containing polyclonal rabbit anti AT antibodies with and without heparin incorporated into the first dimension gel was performed in 50 mM Trisbarbital pH 8.8 buffer.
634
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
Determination of AT III activity/concentration The AT activity was determined on the basis of its ability to inhibit amidolytic activity of thrombin added in definite amount to the system. As the chromogenic substrate, Z-Gly-Pro-Arg-pNA was used (21). Concentration of AT was determined by the EIA method using rabbit polyclonal anti AT antibodies (22). Antithrombin III determination by ELISA Dynatech 96 well microtiter plates were coated with 100 ul of the 11 or 22 nM/ml AT solution. After overnight incubation and three washings, the unoccupied binding sites were blocked with 0.02% gelatin solution for 60 min. After three washings with PBS, 0.05% Tween 20, 100 ul of the first anti AT monoclonal antibody (concentration 0.1 ng/ml to 10 ug/ml) was added and incubated for 90 min at 37°C. After three washings with PBS-Tween 20 solution, 100 ul of the second anti AT monoclonal antibody at a fixed concentration of 2 ug/ml was added and incubated for 90 min at k C. Finally, the swine anti mouse peroxidase labeled IgG was added and after 90 min incubation, the visualization with OPD H„0„ system was performed. The binding curves were computer fitted by nonlinear regression using Hill s equation employing GraphPAD, ISI, USA, computer program. -
RESULTS AND DISCUSSION Three monoclonal antibodies named D8, C5 and B11 have been constructed and isolated and the binding profiles against isolated AT were determined by ELISA technique. Fig. 1 A shows the binding curve of D8 monoclonal antibody while 1 B shows the same experiment performed after the AT coated plate had been first incubated with the C5 monoclonal at a concentration exceeding the saturation level and the excess of the first antibody was washed out. Similarly the binding curves for the interaction of AT with C5 monoclonal antibody is given in Fig. 2 A and with C5 after saturation with D8 in Fig. 2 B. In Table 1 the binding parameter values are given for the binding of monoclonal antibodies to antithrombin III. TABLE 1 The
apparent
(K, app)
dissociation constants of the AT-antibody complexes
Monoclonal
Subclass
antibody
K,
app
(nmolej
D8
IgG
2.4
B11
IgG1 IgG1
13.0 24.0
C5
It follows from the above experiments that D8 and C5 antibodies bind independently and, therefore, that they are directed to the different epitopes on AT molecule. The binding properties of the antibodies were further examined by "native" and SDS PAGE of isolated antithrombin, of human plasma and of human serum by immunoblotting technique. Fig. 3 shows the immunoblots of the PAGE
635
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
1.0
o
en
0.5
0.0 -1
2
1
log
c
(D8) tog/1]
o
01
A. Binding profile of antithrombin III D8 monoclonal antibody interaction obtained with ELISA method. D8 monoclonal interaction B. Binding profile of AT after AT saturation with C5 antibody.
FIGURE 1
-
-
636
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
o
O)
3
2
log
c
(C5) [//g/1]
o O)
3
2
log
c
(C5) [//g/1]
FIGURE 2 A. Binding profile of the antithrombin III C5 monoclonal antibody interaction. B. Binding profile of the AT C5 interaction after AT saturation with D8 monoclonal antibody. -
-
637
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
(p)
and isolated FIGURE 3« Immunoblots of human serum (S), plasma antithrombin III following "native" PAGE developed with D8 and B11 monoclonal antibodies.
(AT)
of isolated AT, serum and plasma developed with D8 and B11 monoclonal antibodies. It follows that while the D8 antibody reacts with the isolated AT as well as with AT of plasma and serum, the B11 antibody reacts predominantly with TAT complexes, so the B1 1 antibody must be directed towards an epitope different than the D8 antibody. The reactivity of the antibodies judged from electrophoretic experiments is summarized in Table 2. TABLE 2
Reactivity
of the anti AT monoclonal antibodies with AT after PAGE and electroblotting
antibody
PAGE "native" serum AT plasma AT TAT AT
D8
X
X
X
B11
X
X
X
C5
X
Monoclonal
SDS PAGE AT
AT(r) X
In conclusion: three different monoclonal antibodies against AT were constructed and isolated of which D8 is apparently the most useful. The detection limit on immunoblots with peroxidase detection is below 1 ug. It is directed towards the sequential epitope present both on native as well as on SDS denatured and reduced molecule which is not influenced by heparin binding. The C5 monoclonal reacts with the isolated AT about 10 times less strongly and no reactivity was observed towards plasma AT on immunoblots. This antibody is apparently directed to the conformational epitope since no reactivity was observed towards SDS denatured AT on immunoblots. The B11 antibody has lower avidity for AT than D8 antibody, it reacts, however, not only with AT in antithrombin complexes plasma and serum but also with thrombin -
638
(TAT). So it appears that the epitope of the AT molecule towards which the B11 antibody is directed is not influenced by the antithrombin-thrombin interaction. A CKNOWLE DGMENTS
Hybridoma 1991.10:633-640. Downloaded from online.liebertpub.com by University of Michigan e-journal package on 12/25/14. For personal use only.
Ms
The authors wish to thank Ms Sedlmaierová, Ms Velebná and Benesová for their skillful technical assistance. REFERENCES
1. 2.
3.
4. 5.
6. 7.
8.
9.. 10. 11.
ABILDGAARD, U. (1968). Highly purified antithrombin III with heparin cofactor activity prepared by disc electrophoresis. ROSENBERG, R.D., and DAMUS, P.S. (1973)- The purification and mechanism of action of human antithrombin-heparin cofactor. J. Biol. Chem. 248, 6490. OWEN, W.G., PENICK, G.D., YEDER, E., and P00LE, B.L. Evidence for an ester bond between thrombin and heparin cofactor. Thromb. Haemostas. 35» 87. ROSENBERG, J.S., McKENNA, P., and ROSENBERG, R.D. (1975). Inhibition of human factor IXa by human antithrombin-heparin cofactor. J. Biol. Chem. 250, 8883. STEAD, N., KAPLAN, A.P., and ROSENBERG, R.D. (1976). Inhibition of activated factor XII by antithrombin-heparin cofactor. J. Biol. Chem. 251, 6481. VENNEROD, A.M., LAAKE, K., SOLBERG, A.K. and STROMLAND, S. (1975)- Inactivation and binding of human plasma kallikrein by antithrombin III and heparin. Thromb. Res. 9, 457« BULLER, H.R., and CATE, J.W. (1989). Acquired antithrombin III deficiency: Laboratory diagnosis, incidence, clinical implications and treatment with antithrombin III concentrate. Am. J. Med. 87 (suppl 3B), 44S. BERESFORD, C.H. (1988). Antithrombin III deficiency. Blood reviews 2, 2 39. PROCHOWNIK, E. (1989). Molecular genetics of inherited antithrombin III deficiencies. Am. J. Med. 87 (suppl 3B)> 15S. HIRSH, J., PIOVELLA, F., and PINT, M. (1989). Congenital antithrombin III deficiency. Am. J. Med. 87 (suppl 3B), 34s. ABILDGAARD, U., LIE, M., and ODEGABD, O.R. (1977). Antithrombin (heparin cofactor) assay with "new" chromogenic substrates (S-2238 and Chromozym- TH) Thromb. Res". 11, 539. HANDELAND, G.F., ABILDGAARD, U., and AASEN, A.O. (1983). Simplified assay for antithrombin III activity using chromogenic peptide substrate. Scand. J. Haematol. 31» 427. SAS, G., PEPPER, D.S., and CASH, J.D.(1975). Investigations on Antithrombin III in normal plasma and serum. Br. J. Haematol.
(19?6).
.
12.
13.
30, 265.
E.J. (198O). Immunological analysis of active and inactive antithrombin III. Br. J. Haematol. 46, 2 77. 15. KNOLLER, S., and SAVION, N. (1989)• Monoclonal antibodies against antithrombin III. Eur. J. Biochem. 180, 319« 16. McKAY, E.J. (1981). A simple two step procedure for the isolation of antithrombin III from biological fluids. Thromb. Res. 21, 37517. KÖHLER, G., and MILSTEIN, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495.
14. McKAY,
18.
FREDRIKSSON, U.B., FAGERSTAM, L.G., COLE, A.W.G., andLUNDGREN, (1986). Protein A-Sepharose CL-4B affinity purification of
T.
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