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Application of sepharose-linked monoclonal antibodies for the immunoradiometric measurement of factor VIII-procoagulant antigen
THROMBOSIS RESEARCH 33; 89-93, 1983 0049-3848183 $3.00 + .OO Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
BRIEF
COMMUNICATION
APPLICATION OF SEPHAROSE-LINKED MONOCLONAL ANTIBODIES FOR THE IMMUNOR4DIOMETRIC MEASUREMENT OF FACTOR VIII-PROCOAGLJLAN'T ANTIGEN
E.C.I. Veer-man,H.V. Stel, J.G. Huisman* and J.A. van Mourik * Department of Blood Coagulation and Department of Molecular Biology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands (Received 1.9.1983; Accepted in revised form 14.10.1983; by Editor J. Stenflo) INTRODUCTION The factor VIII-VWF complex is considered to possess two sets of antigenie determinants, which are correlated with the biological functions exerted by this protein complex. These are the factor VIII-related antigen (VIIIRAg) associated with the Von Willebrand factor moiety, and the factor VIII-procoagulant antigen (VIII&Q) which is associated with the procoagulant moiety of the factor VIII-VWF complex. Until now, several immunological assays for measurement of VIIICAg have been reported (l-7). These assays rely at least in part on the availability of human antisera to VIIICAg which arise in polytransfused hemophilia-A patients or spontaneously in previously healthy persons. A frequently reported method for the measurement of VIIICAg is the twosite immunoradiometric assay (IRMA). In this type of assay, the antigen to be tested is bound to a solid-phase-coated antibody and subsequently detected with an immunopurified radiolabeled antibody. In this paper, we have examined the effect of the solid-phase matrix on the sensitivity of the IRMA for VIIICAg. It appeared that the application of covalent Sepharose-linked antibodies as solid phase enhances the sensitivity of the assay at least tenfold as compared to the sensitivity with the generally used polystyrene-coated antibodies.
MATERIALS AND METHODS Monoclonal antibodies. The production and characterization of the monoclonal antibodies directed to VIIICAg will be described in more detail (Stel et al, in preparation). In the study reported here, we use monoclonal antibodies CLB-CAg A and CLB-CAg 117. CLB-CAg A has been described in more detail (8).
KEY WORDS: IRMA, Factor VIIICAg, Monoclonal antibodies, Sepharose 89
90
FACTOR VIII-PROCOAGULANT
ANTIGEN
Vol. 33, Ho.1
In brief, these two antibodies are directed to the factor-VIII moiety of FVIII-VWP (VIIICAg) as judged by the following criteria: they bind to purified FVIII-VWF, to FVIII-VWF present in normal plasma, but they do not react with the plasma of patients with severe hemophilia A (negative for material cross-reacting with a human antiserum against VIIICAg). Monoclonal antibody CLB-CAg A inhibits VIIIC activity in normal plasma completely and CLB-CAg 117 partly. Purification, radiolabeling and coupling procedure of monoclonal antibodies. Monoclonal antibodies were purified by absorption to protein-A Sepharose (Pharmacia, Uppsala, Sweden) (9). IgG-containing fractions were pooled, neutralized with 0.5 M NaHC03 (pH 8.0), dialyzed overnight against phosphatebuffered saline (PBS), pH 7.2, and stored in small aliquots at -7OOC. One hundred micrograms of antibody were radiolabeled with 0.5 mCi 1251 using the iodogen method (10). The radiolabeled antibodies were stored at -300C and, prior to use, diluted in assay buffer containing 0.05 M Tris, 0.15 M NaCl, 0.05% Tween-29, 3% bovine serum albumin, 0.1% gelatin, 0.14 human IgG, 0.02% NaN3, pH 7.2. This assay buffer appears to be most effective in diminishing aspecific adsorption of the radiolabeled antibodies to Sepharose or polystyrene matrices. Isolation of hlumananti-VIIIC IgG was performed as described by Hellings et al. (6). Immunoglobulins were coupled to CNBr-activated Sepharose-4B (1 mg IgG/ml gel) according to the manufacturers' instructions (Pharmacia). The Sepharose beads were diluted in assay buffer to a final Isolation of specific concentration of 1 mg IgG/lOO ml and stored at 4oC. radio-iodinated human anti-VIIICAg was performed essentially as described by Peake et al. (2). Immunoradiometric assays. The immunoradiometric assay utilizing polystyrene tubes was performed as described elsewhere (6). Briefly, antibodies were coated by incubating plastic tubes with a diluted anti-VIIICAg IgG fraction After subsequent incubation with (20 ug IgG/ml, 0.05 M NaHC03, !H 9.6). serial dilutions of sample, l2 I-labeled anti-VIIICAg IgG was added. After washing, tubes were assayed for bound radioactivity. The imrnunoradiometricassay utilizing Sepharose-linked antibodies was performed as follows: in the first incubation step, samples (100 ~1) were incubated with Sepharose-linked antibodies (400 ~1). Incubation was performed in stoppered polystyrene tubes end-over-end for 20 h at room temperature. Separation of bound-from-free antigen was performed by centrifugation at 3000 g for 2 min. The sedimented Sepharose beads were washed 4 times with 2 ml of In the second incubation step, 400 ~1 raoiolabeled PBS-Tween 20 (0.052). (monoclonal) antibodies (-lo4 cpm) were added to each tube, and incubation was allowed to continue end-over-end for 20 h at room temperature. Sepharose beads were washed again 4 times with PBS-Tween 20 and the bound radioactivity was measured. As negative controls, plasmas were included of two patients with severe hemophilia A, having VIIIC levels ~0.01 U/ml as measured according to the method of Veltkamp et al. (ll), and which were negative for material crossreacting with a human antibody against VIIICAg (CW). RESULTS A comparative study of both IRMA systems (polystyrene tubes versus Sepharose beads) was carried out using different combinations of antibodies As can be deduced from the results presented in Fig. 1 and to VIIICAg. Table I, the Sepharose version of the IRMA yielded a higher sensitivity,
concomitant system that
with uses
91
FACTOR VIII-PROCOAGULANT ANTIGEN
Vol. 33, No.1
higher binding percentages compared to the corresponding The-most striking differences were polystyrene tubes.
117-A*
40A-117' 30 H-H'
A-H'
117-H*
H-117'
1 _L s
values measured in VIIICAg IRMAs, utilizing polystyFig. 1 - Maximal binding rene tube-coated antibodies (m) or Sepharose-linked antibodies (0) as solid H, A, 117, solid-phase antibodies: human anti-VIIICAg, CLB-CAg A and phase. Maximal CLB-CAg 117, respectively. H*, A*, 117*, radiolabeled antibodies. binding was calculated as: radioactivity bound at 1:5 final diluted normal plasma. radioactivity added’ Buffer values ranged between 0.5% (with A* and 117*> and 1.5% (with H*). Plasma of two severe CRM hemophilia-A patients showed no binding of radioactivity.
noted when monoclonal antibodies were assessed for their performances as solid-phase antibody or radiolabeled antibody. In the Sepharose IRMA based on these antibodies, maximal binding values ranged between 25 and 50% and lower limits of detection were between l/1600 and l/2000 U VIIICAg/ml. In contrast, when these antibodies were applied in the polystyrene tube system either as solid-phase antibody or as radiolabeled tracer, no satisfactory assays could be set up. results were improved when an immunopuriSimilarly, fied human radiolabeled antibody was used in the Sepharose version of the assay. It appeared that a batch of immunopurified radiolabeled human antibody, giving a maximal binding of 6% in the polystyrene tube and henceforth not suitable as tracer, performed well in the Sepharose system yielding a maximal binding percentage of 25% (not shown).
FACTOR VIII-PROCOAGULANT
92
ANTIGEN
Vol. 33, No.1
TABLE I Sensitivity of VIIICAg IEMAs, expressed as the final which significantly differed from the buffer value tracer solid
+
dilution of (signal-to-noise
H*
A*
l/160
1 I40
H
l/1600
l/1600
A
l/l600
117
l/1600
normal plasma ratio >2) * 117
phase t
Tube H Sepharose
l/l600
Tube A Sepharose Tube
1I2000
117
Sepharose - , no dose
response.
For
abbreviations,
Furthermore, it was found that used as solid-phase antibody as the antigen could be detected, diolabeled antibody is directed sion of the same antibody.
see
1I2000 legend
to Fig.
1.
if the same monoclonal antibody (A or 117) was well as radiolabeled antibody, no binding of probably because the epitope to which the rawas already occupied by the solid-phase ver-
DISCUSSION Immobilization of antigen in the two-site immunoradiometric assay for VIIICAg is generally accomplished by anti-VIIICAg antibodies that are coated to polystyrene tubes (l-7). For a good performance of the assay in the polystyrene tube system, the source or preparation of these first-phase antibodies as can be deduced from the finding that antidoes not appear to be critical, bodies to VIIICAg, derived from low-titer inhibitor plasmas, perform in this respect as good as high-titer antibodies (4). In contrast, the preparation of the radiolabeled, second-phase antibody appears to be of utmost importance (4) * In the present paper, we have examined the use of another type of matrix to which the first-phase antiboas solid phase, CNBr-activated Sepharose-OB, dies are covalently linked. It appeared that, probably due to a higher density of antibodies provided by this matrix, a higher sensitivity in the VIIICAg compared to that obtained in the assay system based on IRMA was achieved, Furthermore, monoclonal antibodies to VIIICAg of probably polystyrene tubes. low affinity, which apparently did not function in the tube system as tracer or as first-phase antibody, performed excellently in the Sepharose system. this type of solid-phase matrices permitted the use of (radiolabeTherefore, led) antibodies that were not suitable in the polystyrene system and allowed the development of an VIIICAg IRMA that is based exclusively on monoclonal antibodies having different specificity to VIIICAg.
Vol. 33, No.1
FACTOR VIII-PROCOAGULANT
93
ANTIGEN
The sensitivity of the IRMA based on Sepharose-linked antibodies allows detection of VIIICAg levels as low as 5 x 10m4 U/ml, corresponding to 100 pg VIIICAg/ml at the most (12). Although for clinical purposes such sensitivity does not seem to be required at present, it is of utmost importance for other applications, e.g. for studying the in-vitro translation of factor VIII mRNA (13).
REFERENCES I.
Lazarchick, J. and Hoyer, L.W. Immunoradiometric measurement of the fattor VIII procoagulant antigen. J. Clin. Invest. 62, 1048-1052, 1978.
2.
Peake, I.R., Bloom, A.L., Giddings, J.C. and Ludlam, C.A. An immunoradiametric assay for procoagulant factor VIII antigen: results in haemophilia, von Willebrand's disease and fetal plasma and serum. Brit. J. Haematol. 42, 269-281, 1979.
3.
Holmberg, L., Borge, L., Nilsson, R. and Nilsson, I.M. Measurement of antihaemophilic factor antigen (VIII:CAg) with a solid-phase immunoradiametric method based on homologous non-haemophilic antibodies. Scand. J. Haematol. 23, 17-24, 1979. Girma, J.P., Lavergne, J.M., Meyer, D. and Larrieu, M.J. Immunoradiometric assay of factor VIII:coagulant antigen using four human antibodies. Study of 27 cases of haemophilia A. Brit. J. Haematol. 47, 269-282, 1981.
5.
Muller, H.P., van Tilburg, N.H., Bertina, R.M., Terwiel, J.Ph. and Veltkamp, J.J. Immunoradiometric assay of procoagulant factor VIII antigen (VIIICAg). Clin. Chim. Acta 107, 11-19, 1980.
6.
Hellings, J.A., van Leeuwen, F.R., Over, J. and van Mourik, J.A. Immunoradiometric assay of VIII:CAg, a potential tool to detect human VIII:C antibodies. Thromb. Res. 26, 297-302, 1982.
7.
Rotblatt, F., Goodall, A.H., O'Brien, D.P., Rawlings, E., Middleton, S. and Tuddenham, E.G.D. Monoclonal antibodies to human procoagulant factor VIII. J. Lab. Clin. Med. 101, 736-746, 1983.
a.
Stel, H.V., van der Kwast, Th.H. and Veerman, E.C.I. Detection of factor VIII/coagulant antigen in human liver tissue. Nature 303, 530-532, 1983.
9.
Goding, J.W. Conjugation of antibodies with fluorchromes: modification to the standard methods. J. Immunol. Methods 13, 215-226, 1976.
10.
Devare, S.G. and Stephenson, J.R. Biochemical and immunological characterization of major envelope glycoproteins of bovine leukemia virus. J. Virol. 23, 443-444, 1977.
11.
Veltkamp, J.J., Drion, E.F. and Loeliger, E.A. Detection of the carrier state in hereditary coagulation disorders. Thrombos. Diathes. Haemorrh. 19, 297-303, 1968.
12. Hoyer, L.W. The factor VIII complex: structure and function. Blood 58, l-13, 1981. 13.
Bloom, A.L. Benefits of cloning genes for clotting factors. 474-475, 1983.
Nature 303,
BRIEF
COMMUNICATION
APPLICATION OF SEPHAROSE-LINKED MONOCLONAL ANTIBODIES FOR THE IMMUNOR4DIOMETRIC MEASUREMENT OF FACTOR VIII-PROCOAGLJLAN'T ANTIGEN
E.C.I. Veer-man,H.V. Stel, J.G. Huisman* and J.A. van Mourik * Department of Blood Coagulation and Department of Molecular Biology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands (Received 1.9.1983; Accepted in revised form 14.10.1983; by Editor J. Stenflo) INTRODUCTION The factor VIII-VWF complex is considered to possess two sets of antigenie determinants, which are correlated with the biological functions exerted by this protein complex. These are the factor VIII-related antigen (VIIIRAg) associated with the Von Willebrand factor moiety, and the factor VIII-procoagulant antigen (VIII&Q) which is associated with the procoagulant moiety of the factor VIII-VWF complex. Until now, several immunological assays for measurement of VIIICAg have been reported (l-7). These assays rely at least in part on the availability of human antisera to VIIICAg which arise in polytransfused hemophilia-A patients or spontaneously in previously healthy persons. A frequently reported method for the measurement of VIIICAg is the twosite immunoradiometric assay (IRMA). In this type of assay, the antigen to be tested is bound to a solid-phase-coated antibody and subsequently detected with an immunopurified radiolabeled antibody. In this paper, we have examined the effect of the solid-phase matrix on the sensitivity of the IRMA for VIIICAg. It appeared that the application of covalent Sepharose-linked antibodies as solid phase enhances the sensitivity of the assay at least tenfold as compared to the sensitivity with the generally used polystyrene-coated antibodies.
MATERIALS AND METHODS Monoclonal antibodies. The production and characterization of the monoclonal antibodies directed to VIIICAg will be described in more detail (Stel et al, in preparation). In the study reported here, we use monoclonal antibodies CLB-CAg A and CLB-CAg 117. CLB-CAg A has been described in more detail (8).
KEY WORDS: IRMA, Factor VIIICAg, Monoclonal antibodies, Sepharose 89
90
FACTOR VIII-PROCOAGULANT
ANTIGEN
Vol. 33, Ho.1
In brief, these two antibodies are directed to the factor-VIII moiety of FVIII-VWP (VIIICAg) as judged by the following criteria: they bind to purified FVIII-VWF, to FVIII-VWF present in normal plasma, but they do not react with the plasma of patients with severe hemophilia A (negative for material cross-reacting with a human antiserum against VIIICAg). Monoclonal antibody CLB-CAg A inhibits VIIIC activity in normal plasma completely and CLB-CAg 117 partly. Purification, radiolabeling and coupling procedure of monoclonal antibodies. Monoclonal antibodies were purified by absorption to protein-A Sepharose (Pharmacia, Uppsala, Sweden) (9). IgG-containing fractions were pooled, neutralized with 0.5 M NaHC03 (pH 8.0), dialyzed overnight against phosphatebuffered saline (PBS), pH 7.2, and stored in small aliquots at -7OOC. One hundred micrograms of antibody were radiolabeled with 0.5 mCi 1251 using the iodogen method (10). The radiolabeled antibodies were stored at -300C and, prior to use, diluted in assay buffer containing 0.05 M Tris, 0.15 M NaCl, 0.05% Tween-29, 3% bovine serum albumin, 0.1% gelatin, 0.14 human IgG, 0.02% NaN3, pH 7.2. This assay buffer appears to be most effective in diminishing aspecific adsorption of the radiolabeled antibodies to Sepharose or polystyrene matrices. Isolation of hlumananti-VIIIC IgG was performed as described by Hellings et al. (6). Immunoglobulins were coupled to CNBr-activated Sepharose-4B (1 mg IgG/ml gel) according to the manufacturers' instructions (Pharmacia). The Sepharose beads were diluted in assay buffer to a final Isolation of specific concentration of 1 mg IgG/lOO ml and stored at 4oC. radio-iodinated human anti-VIIICAg was performed essentially as described by Peake et al. (2). Immunoradiometric assays. The immunoradiometric assay utilizing polystyrene tubes was performed as described elsewhere (6). Briefly, antibodies were coated by incubating plastic tubes with a diluted anti-VIIICAg IgG fraction After subsequent incubation with (20 ug IgG/ml, 0.05 M NaHC03, !H 9.6). serial dilutions of sample, l2 I-labeled anti-VIIICAg IgG was added. After washing, tubes were assayed for bound radioactivity. The imrnunoradiometricassay utilizing Sepharose-linked antibodies was performed as follows: in the first incubation step, samples (100 ~1) were incubated with Sepharose-linked antibodies (400 ~1). Incubation was performed in stoppered polystyrene tubes end-over-end for 20 h at room temperature. Separation of bound-from-free antigen was performed by centrifugation at 3000 g for 2 min. The sedimented Sepharose beads were washed 4 times with 2 ml of In the second incubation step, 400 ~1 raoiolabeled PBS-Tween 20 (0.052). (monoclonal) antibodies (-lo4 cpm) were added to each tube, and incubation was allowed to continue end-over-end for 20 h at room temperature. Sepharose beads were washed again 4 times with PBS-Tween 20 and the bound radioactivity was measured. As negative controls, plasmas were included of two patients with severe hemophilia A, having VIIIC levels ~0.01 U/ml as measured according to the method of Veltkamp et al. (ll), and which were negative for material crossreacting with a human antibody against VIIICAg (CW). RESULTS A comparative study of both IRMA systems (polystyrene tubes versus Sepharose beads) was carried out using different combinations of antibodies As can be deduced from the results presented in Fig. 1 and to VIIICAg. Table I, the Sepharose version of the IRMA yielded a higher sensitivity,
concomitant system that
with uses
91
FACTOR VIII-PROCOAGULANT ANTIGEN
Vol. 33, No.1
higher binding percentages compared to the corresponding The-most striking differences were polystyrene tubes.
117-A*
40A-117' 30 H-H'
A-H'
117-H*
H-117'
1 _L s
values measured in VIIICAg IRMAs, utilizing polystyFig. 1 - Maximal binding rene tube-coated antibodies (m) or Sepharose-linked antibodies (0) as solid H, A, 117, solid-phase antibodies: human anti-VIIICAg, CLB-CAg A and phase. Maximal CLB-CAg 117, respectively. H*, A*, 117*, radiolabeled antibodies. binding was calculated as: radioactivity bound at 1:5 final diluted normal plasma. radioactivity added’ Buffer values ranged between 0.5% (with A* and 117*> and 1.5% (with H*). Plasma of two severe CRM hemophilia-A patients showed no binding of radioactivity.
noted when monoclonal antibodies were assessed for their performances as solid-phase antibody or radiolabeled antibody. In the Sepharose IRMA based on these antibodies, maximal binding values ranged between 25 and 50% and lower limits of detection were between l/1600 and l/2000 U VIIICAg/ml. In contrast, when these antibodies were applied in the polystyrene tube system either as solid-phase antibody or as radiolabeled tracer, no satisfactory assays could be set up. results were improved when an immunopuriSimilarly, fied human radiolabeled antibody was used in the Sepharose version of the assay. It appeared that a batch of immunopurified radiolabeled human antibody, giving a maximal binding of 6% in the polystyrene tube and henceforth not suitable as tracer, performed well in the Sepharose system yielding a maximal binding percentage of 25% (not shown).
FACTOR VIII-PROCOAGULANT
92
ANTIGEN
Vol. 33, No.1
TABLE I Sensitivity of VIIICAg IEMAs, expressed as the final which significantly differed from the buffer value tracer solid
+
dilution of (signal-to-noise
H*
A*
l/160
1 I40
H
l/1600
l/1600
A
l/l600
117
l/1600
normal plasma ratio >2) * 117
phase t
Tube H Sepharose
l/l600
Tube A Sepharose Tube
1I2000
117
Sepharose - , no dose
response.
For
abbreviations,
Furthermore, it was found that used as solid-phase antibody as the antigen could be detected, diolabeled antibody is directed sion of the same antibody.
see
1I2000 legend
to Fig.
1.
if the same monoclonal antibody (A or 117) was well as radiolabeled antibody, no binding of probably because the epitope to which the rawas already occupied by the solid-phase ver-
DISCUSSION Immobilization of antigen in the two-site immunoradiometric assay for VIIICAg is generally accomplished by anti-VIIICAg antibodies that are coated to polystyrene tubes (l-7). For a good performance of the assay in the polystyrene tube system, the source or preparation of these first-phase antibodies as can be deduced from the finding that antidoes not appear to be critical, bodies to VIIICAg, derived from low-titer inhibitor plasmas, perform in this respect as good as high-titer antibodies (4). In contrast, the preparation of the radiolabeled, second-phase antibody appears to be of utmost importance (4) * In the present paper, we have examined the use of another type of matrix to which the first-phase antiboas solid phase, CNBr-activated Sepharose-OB, dies are covalently linked. It appeared that, probably due to a higher density of antibodies provided by this matrix, a higher sensitivity in the VIIICAg compared to that obtained in the assay system based on IRMA was achieved, Furthermore, monoclonal antibodies to VIIICAg of probably polystyrene tubes. low affinity, which apparently did not function in the tube system as tracer or as first-phase antibody, performed excellently in the Sepharose system. this type of solid-phase matrices permitted the use of (radiolabeTherefore, led) antibodies that were not suitable in the polystyrene system and allowed the development of an VIIICAg IRMA that is based exclusively on monoclonal antibodies having different specificity to VIIICAg.
Vol. 33, No.1
FACTOR VIII-PROCOAGULANT
93
ANTIGEN
The sensitivity of the IRMA based on Sepharose-linked antibodies allows detection of VIIICAg levels as low as 5 x 10m4 U/ml, corresponding to 100 pg VIIICAg/ml at the most (12). Although for clinical purposes such sensitivity does not seem to be required at present, it is of utmost importance for other applications, e.g. for studying the in-vitro translation of factor VIII mRNA (13).
REFERENCES I.
Lazarchick, J. and Hoyer, L.W. Immunoradiometric measurement of the fattor VIII procoagulant antigen. J. Clin. Invest. 62, 1048-1052, 1978.
2.
Peake, I.R., Bloom, A.L., Giddings, J.C. and Ludlam, C.A. An immunoradiametric assay for procoagulant factor VIII antigen: results in haemophilia, von Willebrand's disease and fetal plasma and serum. Brit. J. Haematol. 42, 269-281, 1979.
3.
Holmberg, L., Borge, L., Nilsson, R. and Nilsson, I.M. Measurement of antihaemophilic factor antigen (VIII:CAg) with a solid-phase immunoradiametric method based on homologous non-haemophilic antibodies. Scand. J. Haematol. 23, 17-24, 1979. Girma, J.P., Lavergne, J.M., Meyer, D. and Larrieu, M.J. Immunoradiometric assay of factor VIII:coagulant antigen using four human antibodies. Study of 27 cases of haemophilia A. Brit. J. Haematol. 47, 269-282, 1981.
5.
Muller, H.P., van Tilburg, N.H., Bertina, R.M., Terwiel, J.Ph. and Veltkamp, J.J. Immunoradiometric assay of procoagulant factor VIII antigen (VIIICAg). Clin. Chim. Acta 107, 11-19, 1980.
6.
Hellings, J.A., van Leeuwen, F.R., Over, J. and van Mourik, J.A. Immunoradiometric assay of VIII:CAg, a potential tool to detect human VIII:C antibodies. Thromb. Res. 26, 297-302, 1982.
7.
Rotblatt, F., Goodall, A.H., O'Brien, D.P., Rawlings, E., Middleton, S. and Tuddenham, E.G.D. Monoclonal antibodies to human procoagulant factor VIII. J. Lab. Clin. Med. 101, 736-746, 1983.
a.
Stel, H.V., van der Kwast, Th.H. and Veerman, E.C.I. Detection of factor VIII/coagulant antigen in human liver tissue. Nature 303, 530-532, 1983.
9.
Goding, J.W. Conjugation of antibodies with fluorchromes: modification to the standard methods. J. Immunol. Methods 13, 215-226, 1976.
10.
Devare, S.G. and Stephenson, J.R. Biochemical and immunological characterization of major envelope glycoproteins of bovine leukemia virus. J. Virol. 23, 443-444, 1977.
11.
Veltkamp, J.J., Drion, E.F. and Loeliger, E.A. Detection of the carrier state in hereditary coagulation disorders. Thrombos. Diathes. Haemorrh. 19, 297-303, 1968.
12. Hoyer, L.W. The factor VIII complex: structure and function. Blood 58, l-13, 1981. 13.
Bloom, A.L. Benefits of cloning genes for clotting factors. 474-475, 1983.
Nature 303,