BritichJournal ofHaematology, 1975,31, 5.
A Method for Detecting Factor-VIII Clotting Activity Associated with Factor VIII-related Antigen in Agarose Gels PRUDENCE BIRDAND C. R. RIZZA 0.Yford Hacmophilia Centre, Churchill Hospital, Oxford (Riwivrd 17 F i h a r y 1975; acceptedfor publication
I
March 1975)
SUMMARY.A mcthod for detecting factor-VIII clotting activity in agarosc is described. It is bascd 011 factor VIII promoting coagulation in a mixture of hacmophilic plasma in agarosc which is dctccted by a change in opacity. When this test was used to dctcct factor-VIII clotting activity in a one-dimensional Laurel1 electroimmunoassay for factor VIII-related antigen all the factor-VIII activity was found in thc same position as the factor VIII-related antigen immunoprecipitate. Factor-VIII clotting activity did not appear to bc simply trapped in this immunoprecipitate and therefore it has been concluded that the molecule containing factor-VIII clotting activity carrics factor VIII-related antigen determinants. Factor-VIII clotting activity is absent in the plasma of patients with classical haemophilia but an antigen closely related to factor VIII-activity is present in normal amounts in all cases of haemophilia so far tcsted. This antigen, named factor VIII-related antigen, is assayed by an electroimmuno method using a precipitating rabbit antibody and is absent, or reduced, in the plasma of patients with von Willebrand’s disease (Zimmernian et a/, 1971). There is considcrable uncertainty concerning the relationship of factor-VIII activity to factor VIII-related antigen. Several workers have presented evidence indicating that factorVIII clotting activity and factor VIII-related antigen reside on separate molecules (Zimmerman & Edgington, 1973; Austen, 1974), while others (Hoycr, 1973; Kernoff, 1973) have suggested that factor-VIII clotting activity and factor VIII-related antigen may be on the same molecule. This paper describes a method for detecting factor-VIII clotting activity in an agarose gel, based on the observation that coagulation may be detected in a mixture of agarose and plasma by a cliangc in opacity when fibrin forms in the gel (Bird, 1975). One reason this method was devclopcd was to try to study the relationship of factor-VIII activity to factor VIII-related antigen. We thought that if factor-VIII clotting activity is present on the same molecule as factor VIII-related antigen then it might be possible to detect factor-VIII activity in factor VIII-rclated antigen ininiunoprccipitates formed in agarose gels. MATERIALS AND METHODS
Plasrrrn. Nine volumes ofvenous blood were collected into one volume ofo.13
M
trisodium
Corrcspondcncc: Dr Prudence Bird, Oxford Haemophilia Centre, Churchill Hospital, Headington, Oxford 0x3 7LJ.
6
Prudence Bird and C. R. Rizza
citrate and the plasma separated by cciitrifugation. Normal plasma was used either fresh or after being stored for not more than I week at - 20°C. All haemophilic plasma samples uscd had no detectable factor-VIII activity. Factor-VIII concentrates. Freeze-dried concentrates were prepared by the method of Newman et a1 (1971). The factor-VIII activity in the reconstituted material was approximately 500% normal plasma. Cryoprecipitates were prepared from 20 ml fresh frozen plasma. The plasma was thawed at 4°C and centrifuged at 2700 g for 3 0 min at 4°C. The precipitate was redissolved in 2 ml of citratc/saline (5 parts 0.15 M saline+ I part 0.13 M trisodiuni citrate). Cryoprecipitates were prepared from normal and haemopliilic plasma in the same way. Rabbit arttibody to factor VUI-related aiitigen. Three different antibody preparations wcre uscd. Two antisera, prepared against haemophilic cryoprecipitate, were kindly supplied by Dr P. B. A. Kernoff. They were absorbed with plasma from a patient with severe von Willebrand’s disease whose lcvels of factor-VIII activity and factor VIII-related aiitigcri were both zcro. These antisera did not destroy factor-VIII activity when tested by the method of Rizza & Biggs (1973)at a 1/10dilution. A third rabbit antiserum was obtained from Behringwcrke (Marburg-Lahn). This antiserum contained antibody to factor VIII-related antigen but no detectable antibody to factor-VIII activity. All antisera were heatcd at 56°C for 15 min, mixed with 1/6 volume 0.13 M of trisodium citratc, and absorbed with 1 / 1 0 volume of 25% (v/v) aluminium hydroxide suspension. All antibodies produced only one visible precipitation line with normal plasma when used in the Laurel1 technique. No line was obtained with plasma from a patient with severe von Willebrand’s disease. Rabbit antibody to hiininn a -antitrypsin (Behringwerke, Marburg-Lahn). It reacted with a single protein with al-mobility on immunoelectrophoresis. Htrnian antibody tofactor VIII. A haemophilic plasma containing antibody to factor VIIi at a titre of 1800 Oxford unitslml (Rizza & Biggs, 1973) was used. It was diluted 1/10 in saline and 0.5 nil was used to cover a gel 50 x 20 mni in area. Agarose was obtained from Miles Laboratories Ltd (Research Division). The required concentration was made up fresh for each test in 0.05 M sodium barbitone buffer (pH 8.6) or, for some tests, in 0. I 5 M sodium chloride. Conoentionalfactor-VI~Iclotting assay. Factor-VIII activity was determined in glass tubes by the two-stage method of Biggs & Macfarlane (1966). The results were expressed as a per cent of average normal plasma. Discs cut out of agarose containing factor VIII wcre assayed by a modification of the onestage factor-VIII assay (Biggs, 1972). The discs were imnierscd in 0.1ml of Imidazole buffer (0.05M Imidazole, 0.I M sodium chloride, pH 7.2). Each disc suspensionwas frozen and thawed to try to break up the gel matrix. 0.1ml ofhaemophilic plasma, 0.1ml I/IOOcephalin and 0.1 nil kaolin, 5 mg/ml, were added to the suspension and after 2 min incubation at 37”C, 0.1 ml of 0.025 M calcium chloride was added and the clotting time obtained with each disc was recorded. Zorrnl electrophoresis. 0.6% agarose for electrophoresis was prepared in 0.05 M sodium barbitone buffer (pH 8.6). The molten agarose was poured between two glass slides separated by a U-shaped former to form gels measuring 50 x 70 x I mni. Slots (8 x 2 mnl) were cut in the gels using a double bladed knife and those were filled with 15 p1 samples using ‘Wiretrol’
Factor VIII and Related Antigen in Agarose
7
disposable micropipettes (Drummond Scientific Co., U.S.A.). Electrophoresiswas carried out 011 a cold platten at 4°C at 1.0V/mm for 3 h. Oric-nirrierrsionnl Laiircll cli,crrairrrnrirr.tonssny (Laurcll, 1972). Antiserum was added to 1% agarosc in barbitone buffer at 56°C aiid then the gels poured as described above. The quantity of antiserum added to the gel was adjusted so that the tallest 'rocket' in any plate was approximately 20 m m higli. Wells, 3 inm in diameter, were filled with 5 ptl samples of citratcd plasma, cryoprecipitatesor 1yophilized factor-VIII concentrates. Electrophoresiswas carried out at 4°C at 1.0V/mm for 33 11. Where required the gels were stained for protein by iinmersioii in tannic acid solution (I % w/v in distilled water) for 5 min followed by washing in tap water. Two-dirriensiorial Lnrrrcll clcclroirirnrririoassay (Laurel], 1972).The first dimension zonal electrophoresis was performed as described above. Using a U-shaped former, agarose containing a rabbit antibody to factor VIII-related antigen was poured around the strip containing the elcctrophoresed proteins. The resulting 50x 70x I mm gel was electrophoresed for 16 h at 0.5 V/mm at right angles to the original direction of electrophoresis. The gel was then dried aiid stained with 0.5% Cooniassie Blue solution. DETECTION OF FACTOR VIII CLOTTING ACTIVITY IN AGAROSE GELS
Method A Step I . Factor VIII-containing samples, and appropriate controls where necessary, were clectrophoresed in agarosc gels by one of the above methods aiid then the gel was immcrsed in a bath containing 0.025 M calcium chloride solution at 37°C for 3 min. Step 2 . The excess calcium chloride solution was poured off the gel and the gel was then covered with an agarosc-haemophilic plasma mixture. The agarose-haeniophilic plasma mixture was prepared by mixing an equal volume of haemophilic plasma (0% factor-VIII activity) at'37"C with molten agarose (0.6% w/v in saline) at 56°C. This mixture was prepared immediately before it was required. Approximately 3 nil of the agarose-haemophilic plasina mixture were required to cover a 70 x 50 mm gel. Step 3 . The agarosc gels covered with haemophilic plasma were immediately cooled to 4°C aiid maintained at this temperature. The gels were examined periodically over dark ground illumination for white opaque spots forming in the top agarose-haemophilic plasma layer. Those opacities usually appeared 2-3 h after the addition of the agarose-liaemophilic plasma mixture. The gels were then either immediately photographed over dark ground illuniinations or fixed by immersion in 4% formaldehyde at 4°C for 5 inin aiid photographed at a later date. Method B Early expcriments were carried out using the above basic technique (Method A) but later cxperimcnts were performed using a variation of the technique. 111 the variation, Step I was the same as in Method A. In Step 2, instead of coveriiig the gel with a inolten agarosehaemophilic plasma mixture it was covered with a pre-poured solid agarose gel containing hacmophilic plasma. This latter gel was prepared by mixing an equal volume of haemophilic plasma, warmed briefly to 56"C, with 1.6% agarose in saline at 56°C and immediately pouring it into a U-shaped former. This gave a gel with dimensions 50 x 70 x I mm. This second
8
Prudence Bird and C.R. Rixxa
gel was carefully placed 011 top of the factor VIII-containing gel which had been pre-treated with 0.025 M calcium chloride solution for 3 min at 37°C. The gels were kept at 4'C until fibrin formation commenced, usually about z h, when the upper gel was carefully removcd and both gels were photographed. RESULTS
An example of the method for detecting factor-VIII activity in agarose is shown in Fig I. Normal plasma (factor-VIII activity 90% of normal), a known haemophilic carrier plasma (factor-VIII activity 30% of normal), and plasma from a severely affected haemophiliac were zonally electrophoresed into an agarose gel which was then recalcified, covered with haemopliilic plasma-agarose mixture, and left at 4°C for fibrin to develop, as described in method A. White opaque fibrin bands with mobility intermediatc to C3 and transfcrrin developed for the normal plasma and the plasma of the haemophilic carrier. There was no fibrin formation with corrcsponding mobility for the haemophilic plasma although some fibrin developed close to the origin. This may be due to thc fibrinogen in the electroplioresed haemophilic plasma. The extent of the fibrin bands seems to be related to the factor-VIII content of the starting plasmas. Generally, samplcs with a high factor-VIII content formed fibrin earlier than samples with low factor-VIII levels and this fibrin gradually increased in area with the incubation time. If the temperature of the gel was not kept low, generalized clotting of the haemophilic plasma in the agarose top layer occurred making clear factor-VIII detection inipossiblc.
of the Mcthod The specificity of the method for factor-VIII activity is shown in the previous experiment by the absence of fibrin formation when a haemophilic plasma was tested. Additional evidence for the specificity of this test for factor-VIII activity has been found by using an antibody to factor VIII, which had developed in a haemophiliac, to inhibit factor-VIII clotting activity in the gel. In this experiment, normal plasma was electrophoresed into an agarose gel from two troughs and the gel then cut into two. One half was covered with a I in 10 dilution of haeniophilic plasma containing a potent antibody to factor-VIII activity, while the sccoiid was covered with a I in 10dilution of plasma from a haemophiliac known not to have antibodies to factor VIII. Both strips were incubated for 30 niin at 37"C, the surplus haemophilic plasma was then washed off with saline and then the haemophilic plasma-agarose mixturc poured on. The gel which had been pre-treated with antibody-containing haeniophilic plasma produced no fibrin opacity in the top haemophilic plasma-agarose layer whereas the gel treated with haemophilic plasma free of antibody formed fibrin opacities in the normal way. An attempt was also made to determine the position of factor-VIII activity in an agarose gel by a conventional tube assay for factor-VIII clotting activity, so that this could be conipared with the position of fibrin formation in the haemophilic plasma agarose test. In order to do this, a factor-VIII concentrate was electrophoresed into a gel from three identical troughs and the gel was then cut into three strips each containing the electrophoresed concentrate. One strip was treated with calcium chloride solution and then covered with agarosehaemophilic plasma mixture and the fibrin band which developed after z h incubation at Specijicity
Factor VIII and Related Antigen in Agarose
FIG I . This dark-ground photograph shows the position of factor VIII after electrophoresis in agarosc as measured by the formation of fibrin in the haemophilic plasma-agarose factor-VIII clotting test. Upper strip: normal plasma; centre strip: carrier plasma; lower strip: haemophilic plasma.
(Facing p 8)
Prudence Bird and C.R. Rizza
FIG 2. Specificity of haemophilic plasma-agarose factor-VIII clotting test. A factor-VIII concentrate was electrophoresed into three parallel strips in a 0.6% agarose gel. The factor VIII was detected in each strip by a different method to show the specificity of the haemophilic plasma-agarose factor-VIII clotting test. (a) A two-dimensional electroimrnunoassay for factor VIII-related antigen. (b) The thin layer factor-VII1 clotting test showing white spots of fibrin formation in the position of factor VIII. (c) Clotting times in seconds for 2 mm discs cut from a third strip with their centres at the distances indicated from the origin.
FIG 3. A one-dimensional Laurell electroirnmunoassay for factor VIII-related antigen. One half (a) was developed with tannic acid and the other (b) with haemophilic plasma-agarose. The wells contained dilutions of a factor-VIII concentrate (A), and a haemophilic cryoprecipitate (B).
FIG 4. (a) A onedimensional Laurell electroinimuiioassay for factor VIIIrelated antigen. (The wells contained dilutions of normal cryoprecipitate (A) and a haemophilic cryoprecipitate (B).) (b) A haemophilic plasmaagarose gel which had beeii left in contact with gel (a) until fibrin formed and then separated from it.
Fuctor Vlll and Related Antigen in Agarose
Prudence Bird and C.R. Rizza
FIG 5 . Experiment to show that factor VIII is not sterically trapped by immunoprecipitates. The wells in each gel contained dilutioiis of a normal cryoprecipitate (A), haemophilic cryoprecipitate (B), and a factor-VIII concentrate (C). (a) This Laurell electroimmunoassay gel contained rabbit antiserum to factor VIII-related antigen. (b) This Laurell electroimmunoassay contained rabbit antiserum to a,antitrypsin. After electrophoresis, both gels were covered with a layer of haemophilic plasma-agarose mixture.
Factor VIII and Related Antigen in Agarose
9
4’C is shown in Fig 2(b). Discs of agarose, each 2 mm in diameter, were removed from the second strip with their centres 2, 6, 10, 14, etc., mm from the origin. Each disc was immersed in 0.1ml glyoxaline buffer in a glass tube, for assay by the modified one-stage factor VIII assay described in the Methods. The clotting time obtained with each disc was recorded and is shown in Fig 2(c). The maximuni reduction of clotting time in the factor-VIII assay system was obtained with those discs which had been cut from tlie agarose at the same distance from the origin as the fibrin formation in the parallel strip (Fig 2b). The reduction is small, possibly because only a small quantity of factor VIII in the discs was available, but it nevertheless suggests that the area of fibrin formation is the area which contains most factor-VIII activity. The third agarose strip was used as tlie first dimension in ,a two-dimensional Laurell electroimmunoassay for factor VIII-related antigen. Fig 2(a) shows the position of factor VIIIrelated antigen in this test. Factor VIII-related antigen closely resembles factor-VIII activity in mobility as measured by the above methods.
The Rclatiotzship of Factor-VIII Clotting Activity to Factor VIII-Related Antigeri hi these experiments, the position of factor-VIII clotting activity was detected by the haemophilic plasma-agarose method after precipitation of factor VIII-related antigen in a one-dimensional Laurell electroimmunoassay. Fig 3 shows two halves of a one-dimensional Laurell electroimmunoassay for factor VIII-related antigen. The I yo agarose contained 6% Rehringwerke antiserum to factor VIII-related antigen and the wells in each half were filled with dilutions of a factor-VIII concentrate or haeniophilic cryoprecipitate. After electrophoresis o m of tlie two halves of the gel was immersed in 1% tannic acid to develop the factor VIII-related antigen immunoprecipitatcs (Fig 3a) aiid over the other half was poured a liaemophilic plasma-agarose mixture to detect the position of factor-VIII clotting activity (Fig 3b). The fibrin spots which developed in relation to tlie various dilutions of the factorVIII concentrate in this second half formed typical ‘rocket’shapes which corresponded in outline and lieiglit with the ininiunoprecipitates of tlie factor VIII-related antigen in the other half of the gel. There was no fibrin formation around the factor VIII-related antigen precipitate of the haemophilic cryoprecipitate which can be seen faintly in the gel covered with haemopliilic plasma-agarose as well as in Fig 3(a). Normal cryoprccipitates (see for example Fig 4) aiid normal plasma in other experinients gave the same results as the factor-VIII conce~itrateshown here. Concentrated preparations of factor VIII have been used in this work to show tlie identical height and shape of the fibrin aiid iinniunoprccipitate ‘rockets’ because the factor VIII-related antigen immunoprecipitates obtained with normal and hacmophilic plasmas were difficult to record clearly. This appears to be associated with the short length of time used for the electroimmunoassay which is needed if factor-VIII clotting activity is to be measured. Overnight electrophoresis, which is the standard practice for assaying factor VIII-related antigen, produces good immunoprecipitates but very little factor-VIII activity can then be detected in the precipitates. To check that the fibrin spots corresponded exactly in outline aiid height with the corresponding ininiuiioprccipitates of factor VIII-related antigen, a variation 011 the original factor VIII-agarosc clotting test was developed (method B). In this method, the liaeniophilic plasnia-agarose layer can be liftcd off when fibrin formation conimenccs so that the position of tlie fibrin formation in tlie haemophilic agarose can be directly compared with thc position
I0
Prudence Bird atrd C.R.Rirzo
of the immunoprecipitatesin the underlying agarose used for the Laurell electroimmunoassay. This has been done for the gel shown in Fig 4(a). The wells in the one-dimensional Laurell agarose, which contained 6% Behringwerke antiserum to factor VIII-related antigen, were filled with dilutions of a normal cryoprecipitate and a haemophilic cryoprecipitate. After electrophoresis the gel was covered with a preformed liaemophilic plasma-agarose gel and when fibrin formation commenced this top gel was removed and both gels were photographed side by side. Fig 4(a) shows the factor VIII-related antigen immunoprecipitates. The faint second line that may be seen beyond the main precipitate (here and in Fig 5 ) results from uneven running of the cryoprecipitate through the agarose and not to a second antibody valency. Fig 4(b) shows the fibrin formation in the upper haemophilic-agarose layer. The heights of the fibrin ‘rockets’ for the normal cryoprecipitate dilutions correspond with that of their immunoprecipitates. Once again the haemophilic cryoprecipitate showed only factor VIII-related antigen and no fibrin formation. To eliminate the possibility that the factor-VIII activity might be simply trapped in the factor VIII-related antigen immunoprecipitate, the one-dimensional Laurell electroimmunoassays were repeated using antiserum in the agarose against plasma proteins other than factor VIII-related antigen. If factor-VIII activity was found to be associated also with these other immunoprecipitates a non-specific trapping mechanism would be suspected. Anti-a, -antitrypsin was chosen for this experiment since a -antitrypsin has a faster electrophoreticmobility than factor-VIII activity, making it necessary for factor-VIII activity to migrate through the a -antitrypsin imniunoprecipitate. One half of the agarose gel shown in Fig 5 contained 7% antiserum to factor VIII-related antigen whilst the other half contained 20% antiserum to a,-antitrypsin. The wells contained cryoprecipitates from normal and haemophilic plasmas and a factor-VIII concentrate. All these samples contained a -antitrypsin. The one-dimensional electroimmunoassay was developed for factor-VIII activity. All the fibrin in the agarose gel containing anti-factor VIIIrelated antigen formed along the immune precipitates whilst in the agarose gel containing anti-a, -antitrypsin, the fibrin formed beyond the immunoprecipitates. This shows that factor-VIII activity migration was not trapped by antie,-antitrypsin immune precipitate. It is therefore unlikely that the anti-factor VIII-related antigen immune precipitate was now specifically trapping factor-VIII activity. It is possible that the rabbit antiserum to factor VIII-related antigen was not monospecific. There may have been one antibody population recognizing and precipitating factor VIIIrelated antigen and another antibody population recognizing factor-VIII activity protein but forming immune complexes which may not be detected by precipitation techniques. To eliminate this possibility, the experiments with the electroimmunoassay were repeated with three different antisera to factor VIII-related antigen in the agarose. The relative quantity of antibodies of different specificities would be expected to vary in the different antisera but in all cases fibrin formation in the superimposed haemophilic plasma-agarose gel coincided with the factor VIII-related antigen immunoprecipitates.
,
DISCUSSION Using an agarose gel overlay containing haemophilic plasma it is now possible to detect
Factor VIII and Rdated Antigen in Agarose
I1
factor-VIII clotting activity directly after it has been electrophoresed into an agarose gel and it is hoped that this test may be adapted to detect factor VIII in other thin layer separating techniques. The specificity of this test for factor-VIII activity has been checked in several ways and whenever possible haemophilic samples from severely affected haemophiliacs have been included as negative controls. This new test is not quantitative for factor VIII although the speed and extent of fibrin formation in the haemophilic plasma agarose seems to be related to the level of factor-VIII activity in the agarose. It was capable of detecting the factor-VIII clotting activity contained in a 15 ~1 sample of plasma and a factor-VIII level of 25% of normal. This thin layer test has allowed us to relate the clotting activity of factor VIII to immunoprecipitates of factor VIII-related antigen. This required a rabbit antibody to factor VIIIrelated antigen which would form precipitates but which did not neutralize factor-VIII activity. The present studies show that all the factor-VIII clotting activity detectable by this method is found in the same place in the agarose gel as the factor VIII-related antigen immunoprecipitates. In these electroimmunoassays,fibrin formation in the haemophilic plasma agarose overlay occurred sooner than when zonal electrophoresis was used, suggesting that factor VIII was concentrated along the immunoprecipitate. This suggests that much smaller quantities of factor-VIII clotting activity may be detected by this method compared to zonal electrophoresis. Control experiments have shown that it is unlikely that factor-VIII activity was being concentrated in this precipitate by a trapping mechanism or that there was an antibody population to factor-VIII activity complexing factor-VIII activity which was distinct to that for factor VIII-related antigen. We are therefore left with the tentative conclusion that the molecule containing factorVIII activity was associated specifically with the factor VIII-related antigen immunoprecipitatc. This suggests that factor-VIII clotting activity and factor VIII-related antigen determinant(s) are present on the same n~olecule. This conclusion is not compatible with the data of Zimmerman & Edgington (1973), who used human antibody to factor VIII attached to beads and found that it removed factor-VIII activity but not factor VIII-related antigen from normal plasma. If, however, only a small percentage of factor VIII-related antigen molecules also express factor-VIII clotting activity this discrepancy could be explained. It is impossible to draw any firm conclusions at this time on the exact relationship of factorVIII activity and factor VIII-related antigen but the work we present here provides evidence to suggest that these activities are very closely related. ACKNOWLEDGMENTS
We should like to thank Dr Rosemary Biggs and Dr D. E. G. Austen for helpful discussion and Mr R. Borrett for his help with the photography. A grant from Action for the Crippled Child is gratefully acknowledged. REFERENCES BIGGS,R. & MACFARLANB, R.G. (1966) Treatment of weight and its aggregation. British Jorrmal .f Haernophilia and Other Coagirlation Disorders, p 3 50. Haerriafology, 27, 89. Blackwell Scientific Publications, Oxford.
AUSTEN, D.E.G. (1974) Factor VIII of small molecular
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Prudence Bird and C . R. Rixza
BIGGS,R. (1972)Human Blood Coagulation, Haemostasis and Thrombosis, p 614.Blackwell Scientific Publications, Oxford. BIRD,P. (1975)Coagulation in an agarose gel and its application to the detection and measurement of factor VIII antibodies. BritishJournal of Haematology, 29s 329. HOYER, L.W. (1973)Specificity of precipitating antibodies in immunological identification of antihaemophilic factor. Nature: New Biology, 245, 49. KERNOFF, P.B.A. (1973)Affinity offactor VIII clotting activity for antigen detectable immunologically. Nature: New Biology, 244, 148. LAURELL, C.-B. (1972) Electroimmunoassay. Scandinavian Journal of Clinical and Laboratory Investigatiorr, 29, SUppl. 124, p 21.
NEWMAN J., JOHNSON,A.J., KARPATKIN, M.H. & PUSZKIN, S . (1971)Methods for the production of clinically effective intermediate and high-purity factor-VIII concentrates. British Joirrnal of Haeniotology, 21, I . RIZZA,C.R. & BIGGS,R. (1973) The treatment of patients who have factor-VIII antibodies. Britisfi Journal of Haetnatology, 24, 65. ZIMMERMAN, T.S. & EDGINGTON, T.S. (1973)Factor VIII coagulant activity and factor VIII-like antigen: independent molecular entities. ]orrrnal of Expcrimental Medicine, 138, 101s. ZIMMERMAN, T.S., RATNOFF, O.D. & POWELL, A.E. (1971) Immunologic differentiation of classic hemophilia (factor VIII deficiency) and von Willebrand’s disease.Journal of Clinical Investigation, 50, 244.
A Method for Detecting Factor-VIII Clotting Activity Associated with Factor VIII-related Antigen in Agarose Gels PRUDENCE BIRDAND C. R. RIZZA 0.Yford Hacmophilia Centre, Churchill Hospital, Oxford (Riwivrd 17 F i h a r y 1975; acceptedfor publication
I
March 1975)
SUMMARY.A mcthod for detecting factor-VIII clotting activity in agarosc is described. It is bascd 011 factor VIII promoting coagulation in a mixture of hacmophilic plasma in agarosc which is dctccted by a change in opacity. When this test was used to dctcct factor-VIII clotting activity in a one-dimensional Laurel1 electroimmunoassay for factor VIII-related antigen all the factor-VIII activity was found in thc same position as the factor VIII-related antigen immunoprecipitate. Factor-VIII clotting activity did not appear to bc simply trapped in this immunoprecipitate and therefore it has been concluded that the molecule containing factor-VIII clotting activity carrics factor VIII-related antigen determinants. Factor-VIII clotting activity is absent in the plasma of patients with classical haemophilia but an antigen closely related to factor VIII-activity is present in normal amounts in all cases of haemophilia so far tcsted. This antigen, named factor VIII-related antigen, is assayed by an electroimmuno method using a precipitating rabbit antibody and is absent, or reduced, in the plasma of patients with von Willebrand’s disease (Zimmernian et a/, 1971). There is considcrable uncertainty concerning the relationship of factor-VIII activity to factor VIII-related antigen. Several workers have presented evidence indicating that factorVIII clotting activity and factor VIII-related antigen reside on separate molecules (Zimmerman & Edgington, 1973; Austen, 1974), while others (Hoycr, 1973; Kernoff, 1973) have suggested that factor-VIII clotting activity and factor VIII-related antigen may be on the same molecule. This paper describes a method for detecting factor-VIII clotting activity in an agarose gel, based on the observation that coagulation may be detected in a mixture of agarose and plasma by a cliangc in opacity when fibrin forms in the gel (Bird, 1975). One reason this method was devclopcd was to try to study the relationship of factor-VIII activity to factor VIII-related antigen. We thought that if factor-VIII clotting activity is present on the same molecule as factor VIII-related antigen then it might be possible to detect factor-VIII activity in factor VIII-rclated antigen ininiunoprccipitates formed in agarose gels. MATERIALS AND METHODS
Plasrrrn. Nine volumes ofvenous blood were collected into one volume ofo.13
M
trisodium
Corrcspondcncc: Dr Prudence Bird, Oxford Haemophilia Centre, Churchill Hospital, Headington, Oxford 0x3 7LJ.
6
Prudence Bird and C. R. Rizza
citrate and the plasma separated by cciitrifugation. Normal plasma was used either fresh or after being stored for not more than I week at - 20°C. All haemophilic plasma samples uscd had no detectable factor-VIII activity. Factor-VIII concentrates. Freeze-dried concentrates were prepared by the method of Newman et a1 (1971). The factor-VIII activity in the reconstituted material was approximately 500% normal plasma. Cryoprecipitates were prepared from 20 ml fresh frozen plasma. The plasma was thawed at 4°C and centrifuged at 2700 g for 3 0 min at 4°C. The precipitate was redissolved in 2 ml of citratc/saline (5 parts 0.15 M saline+ I part 0.13 M trisodiuni citrate). Cryoprecipitates were prepared from normal and haemopliilic plasma in the same way. Rabbit arttibody to factor VUI-related aiitigen. Three different antibody preparations wcre uscd. Two antisera, prepared against haemophilic cryoprecipitate, were kindly supplied by Dr P. B. A. Kernoff. They were absorbed with plasma from a patient with severe von Willebrand’s disease whose lcvels of factor-VIII activity and factor VIII-related aiitigcri were both zcro. These antisera did not destroy factor-VIII activity when tested by the method of Rizza & Biggs (1973)at a 1/10dilution. A third rabbit antiserum was obtained from Behringwcrke (Marburg-Lahn). This antiserum contained antibody to factor VIII-related antigen but no detectable antibody to factor-VIII activity. All antisera were heatcd at 56°C for 15 min, mixed with 1/6 volume 0.13 M of trisodium citratc, and absorbed with 1 / 1 0 volume of 25% (v/v) aluminium hydroxide suspension. All antibodies produced only one visible precipitation line with normal plasma when used in the Laurel1 technique. No line was obtained with plasma from a patient with severe von Willebrand’s disease. Rabbit antibody to hiininn a -antitrypsin (Behringwerke, Marburg-Lahn). It reacted with a single protein with al-mobility on immunoelectrophoresis. Htrnian antibody tofactor VIII. A haemophilic plasma containing antibody to factor VIIi at a titre of 1800 Oxford unitslml (Rizza & Biggs, 1973) was used. It was diluted 1/10 in saline and 0.5 nil was used to cover a gel 50 x 20 mni in area. Agarose was obtained from Miles Laboratories Ltd (Research Division). The required concentration was made up fresh for each test in 0.05 M sodium barbitone buffer (pH 8.6) or, for some tests, in 0. I 5 M sodium chloride. Conoentionalfactor-VI~Iclotting assay. Factor-VIII activity was determined in glass tubes by the two-stage method of Biggs & Macfarlane (1966). The results were expressed as a per cent of average normal plasma. Discs cut out of agarose containing factor VIII wcre assayed by a modification of the onestage factor-VIII assay (Biggs, 1972). The discs were imnierscd in 0.1ml of Imidazole buffer (0.05M Imidazole, 0.I M sodium chloride, pH 7.2). Each disc suspensionwas frozen and thawed to try to break up the gel matrix. 0.1ml ofhaemophilic plasma, 0.1ml I/IOOcephalin and 0.1 nil kaolin, 5 mg/ml, were added to the suspension and after 2 min incubation at 37”C, 0.1 ml of 0.025 M calcium chloride was added and the clotting time obtained with each disc was recorded. Zorrnl electrophoresis. 0.6% agarose for electrophoresis was prepared in 0.05 M sodium barbitone buffer (pH 8.6). The molten agarose was poured between two glass slides separated by a U-shaped former to form gels measuring 50 x 70 x I mni. Slots (8 x 2 mnl) were cut in the gels using a double bladed knife and those were filled with 15 p1 samples using ‘Wiretrol’
Factor VIII and Related Antigen in Agarose
7
disposable micropipettes (Drummond Scientific Co., U.S.A.). Electrophoresiswas carried out 011 a cold platten at 4°C at 1.0V/mm for 3 h. Oric-nirrierrsionnl Laiircll cli,crrairrrnrirr.tonssny (Laurcll, 1972). Antiserum was added to 1% agarosc in barbitone buffer at 56°C aiid then the gels poured as described above. The quantity of antiserum added to the gel was adjusted so that the tallest 'rocket' in any plate was approximately 20 m m higli. Wells, 3 inm in diameter, were filled with 5 ptl samples of citratcd plasma, cryoprecipitatesor 1yophilized factor-VIII concentrates. Electrophoresiswas carried out at 4°C at 1.0V/mm for 33 11. Where required the gels were stained for protein by iinmersioii in tannic acid solution (I % w/v in distilled water) for 5 min followed by washing in tap water. Two-dirriensiorial Lnrrrcll clcclroirirnrririoassay (Laurel], 1972).The first dimension zonal electrophoresis was performed as described above. Using a U-shaped former, agarose containing a rabbit antibody to factor VIII-related antigen was poured around the strip containing the elcctrophoresed proteins. The resulting 50x 70x I mm gel was electrophoresed for 16 h at 0.5 V/mm at right angles to the original direction of electrophoresis. The gel was then dried aiid stained with 0.5% Cooniassie Blue solution. DETECTION OF FACTOR VIII CLOTTING ACTIVITY IN AGAROSE GELS
Method A Step I . Factor VIII-containing samples, and appropriate controls where necessary, were clectrophoresed in agarosc gels by one of the above methods aiid then the gel was immcrsed in a bath containing 0.025 M calcium chloride solution at 37°C for 3 min. Step 2 . The excess calcium chloride solution was poured off the gel and the gel was then covered with an agarosc-haemophilic plasma mixture. The agarose-haeniophilic plasma mixture was prepared by mixing an equal volume of haemophilic plasma (0% factor-VIII activity) at'37"C with molten agarose (0.6% w/v in saline) at 56°C. This mixture was prepared immediately before it was required. Approximately 3 nil of the agarose-haemophilic plasina mixture were required to cover a 70 x 50 mm gel. Step 3 . The agarosc gels covered with haemophilic plasma were immediately cooled to 4°C aiid maintained at this temperature. The gels were examined periodically over dark ground illumination for white opaque spots forming in the top agarose-haemophilic plasma layer. Those opacities usually appeared 2-3 h after the addition of the agarose-liaemophilic plasma mixture. The gels were then either immediately photographed over dark ground illuniinations or fixed by immersion in 4% formaldehyde at 4°C for 5 inin aiid photographed at a later date. Method B Early expcriments were carried out using the above basic technique (Method A) but later cxperimcnts were performed using a variation of the technique. 111 the variation, Step I was the same as in Method A. In Step 2, instead of coveriiig the gel with a inolten agarosehaemophilic plasma mixture it was covered with a pre-poured solid agarose gel containing hacmophilic plasma. This latter gel was prepared by mixing an equal volume of haemophilic plasma, warmed briefly to 56"C, with 1.6% agarose in saline at 56°C and immediately pouring it into a U-shaped former. This gave a gel with dimensions 50 x 70 x I mm. This second
8
Prudence Bird and C.R. Rixxa
gel was carefully placed 011 top of the factor VIII-containing gel which had been pre-treated with 0.025 M calcium chloride solution for 3 min at 37°C. The gels were kept at 4'C until fibrin formation commenced, usually about z h, when the upper gel was carefully removcd and both gels were photographed. RESULTS
An example of the method for detecting factor-VIII activity in agarose is shown in Fig I. Normal plasma (factor-VIII activity 90% of normal), a known haemophilic carrier plasma (factor-VIII activity 30% of normal), and plasma from a severely affected haemophiliac were zonally electrophoresed into an agarose gel which was then recalcified, covered with haemopliilic plasma-agarose mixture, and left at 4°C for fibrin to develop, as described in method A. White opaque fibrin bands with mobility intermediatc to C3 and transfcrrin developed for the normal plasma and the plasma of the haemophilic carrier. There was no fibrin formation with corrcsponding mobility for the haemophilic plasma although some fibrin developed close to the origin. This may be due to thc fibrinogen in the electroplioresed haemophilic plasma. The extent of the fibrin bands seems to be related to the factor-VIII content of the starting plasmas. Generally, samplcs with a high factor-VIII content formed fibrin earlier than samples with low factor-VIII levels and this fibrin gradually increased in area with the incubation time. If the temperature of the gel was not kept low, generalized clotting of the haemophilic plasma in the agarose top layer occurred making clear factor-VIII detection inipossiblc.
of the Mcthod The specificity of the method for factor-VIII activity is shown in the previous experiment by the absence of fibrin formation when a haemophilic plasma was tested. Additional evidence for the specificity of this test for factor-VIII activity has been found by using an antibody to factor VIII, which had developed in a haemophiliac, to inhibit factor-VIII clotting activity in the gel. In this experiment, normal plasma was electrophoresed into an agarose gel from two troughs and the gel then cut into two. One half was covered with a I in 10 dilution of haeniophilic plasma containing a potent antibody to factor-VIII activity, while the sccoiid was covered with a I in 10dilution of plasma from a haemophiliac known not to have antibodies to factor VIII. Both strips were incubated for 30 niin at 37"C, the surplus haemophilic plasma was then washed off with saline and then the haemophilic plasma-agarose mixturc poured on. The gel which had been pre-treated with antibody-containing haeniophilic plasma produced no fibrin opacity in the top haemophilic plasma-agarose layer whereas the gel treated with haemophilic plasma free of antibody formed fibrin opacities in the normal way. An attempt was also made to determine the position of factor-VIII activity in an agarose gel by a conventional tube assay for factor-VIII clotting activity, so that this could be conipared with the position of fibrin formation in the haemophilic plasma agarose test. In order to do this, a factor-VIII concentrate was electrophoresed into a gel from three identical troughs and the gel was then cut into three strips each containing the electrophoresed concentrate. One strip was treated with calcium chloride solution and then covered with agarosehaemophilic plasma mixture and the fibrin band which developed after z h incubation at Specijicity
Factor VIII and Related Antigen in Agarose
FIG I . This dark-ground photograph shows the position of factor VIII after electrophoresis in agarosc as measured by the formation of fibrin in the haemophilic plasma-agarose factor-VIII clotting test. Upper strip: normal plasma; centre strip: carrier plasma; lower strip: haemophilic plasma.
(Facing p 8)
Prudence Bird and C.R. Rizza
FIG 2. Specificity of haemophilic plasma-agarose factor-VIII clotting test. A factor-VIII concentrate was electrophoresed into three parallel strips in a 0.6% agarose gel. The factor VIII was detected in each strip by a different method to show the specificity of the haemophilic plasma-agarose factor-VIII clotting test. (a) A two-dimensional electroimrnunoassay for factor VIII-related antigen. (b) The thin layer factor-VII1 clotting test showing white spots of fibrin formation in the position of factor VIII. (c) Clotting times in seconds for 2 mm discs cut from a third strip with their centres at the distances indicated from the origin.
FIG 3. A one-dimensional Laurell electroirnmunoassay for factor VIII-related antigen. One half (a) was developed with tannic acid and the other (b) with haemophilic plasma-agarose. The wells contained dilutions of a factor-VIII concentrate (A), and a haemophilic cryoprecipitate (B).
FIG 4. (a) A onedimensional Laurell electroinimuiioassay for factor VIIIrelated antigen. (The wells contained dilutions of normal cryoprecipitate (A) and a haemophilic cryoprecipitate (B).) (b) A haemophilic plasmaagarose gel which had beeii left in contact with gel (a) until fibrin formed and then separated from it.
Fuctor Vlll and Related Antigen in Agarose
Prudence Bird and C.R. Rizza
FIG 5 . Experiment to show that factor VIII is not sterically trapped by immunoprecipitates. The wells in each gel contained dilutioiis of a normal cryoprecipitate (A), haemophilic cryoprecipitate (B), and a factor-VIII concentrate (C). (a) This Laurell electroimmunoassay gel contained rabbit antiserum to factor VIII-related antigen. (b) This Laurell electroimmunoassay contained rabbit antiserum to a,antitrypsin. After electrophoresis, both gels were covered with a layer of haemophilic plasma-agarose mixture.
Factor VIII and Related Antigen in Agarose
9
4’C is shown in Fig 2(b). Discs of agarose, each 2 mm in diameter, were removed from the second strip with their centres 2, 6, 10, 14, etc., mm from the origin. Each disc was immersed in 0.1ml glyoxaline buffer in a glass tube, for assay by the modified one-stage factor VIII assay described in the Methods. The clotting time obtained with each disc was recorded and is shown in Fig 2(c). The maximuni reduction of clotting time in the factor-VIII assay system was obtained with those discs which had been cut from tlie agarose at the same distance from the origin as the fibrin formation in the parallel strip (Fig 2b). The reduction is small, possibly because only a small quantity of factor VIII in the discs was available, but it nevertheless suggests that the area of fibrin formation is the area which contains most factor-VIII activity. The third agarose strip was used as tlie first dimension in ,a two-dimensional Laurell electroimmunoassay for factor VIII-related antigen. Fig 2(a) shows the position of factor VIIIrelated antigen in this test. Factor VIII-related antigen closely resembles factor-VIII activity in mobility as measured by the above methods.
The Rclatiotzship of Factor-VIII Clotting Activity to Factor VIII-Related Antigeri hi these experiments, the position of factor-VIII clotting activity was detected by the haemophilic plasma-agarose method after precipitation of factor VIII-related antigen in a one-dimensional Laurell electroimmunoassay. Fig 3 shows two halves of a one-dimensional Laurell electroimmunoassay for factor VIII-related antigen. The I yo agarose contained 6% Rehringwerke antiserum to factor VIII-related antigen and the wells in each half were filled with dilutions of a factor-VIII concentrate or haeniophilic cryoprecipitate. After electrophoresis o m of tlie two halves of the gel was immersed in 1% tannic acid to develop the factor VIII-related antigen immunoprecipitatcs (Fig 3a) aiid over the other half was poured a liaemophilic plasma-agarose mixture to detect the position of factor-VIII clotting activity (Fig 3b). The fibrin spots which developed in relation to tlie various dilutions of the factorVIII concentrate in this second half formed typical ‘rocket’shapes which corresponded in outline and lieiglit with the ininiunoprecipitates of tlie factor VIII-related antigen in the other half of the gel. There was no fibrin formation around the factor VIII-related antigen precipitate of the haemophilic cryoprecipitate which can be seen faintly in the gel covered with haemopliilic plasma-agarose as well as in Fig 3(a). Normal cryoprccipitates (see for example Fig 4) aiid normal plasma in other experinients gave the same results as the factor-VIII conce~itrateshown here. Concentrated preparations of factor VIII have been used in this work to show tlie identical height and shape of the fibrin aiid iinniunoprccipitate ‘rockets’ because the factor VIII-related antigen immunoprecipitates obtained with normal and hacmophilic plasmas were difficult to record clearly. This appears to be associated with the short length of time used for the electroimmunoassay which is needed if factor-VIII clotting activity is to be measured. Overnight electrophoresis, which is the standard practice for assaying factor VIII-related antigen, produces good immunoprecipitates but very little factor-VIII activity can then be detected in the precipitates. To check that the fibrin spots corresponded exactly in outline aiid height with the corresponding ininiuiioprccipitates of factor VIII-related antigen, a variation 011 the original factor VIII-agarosc clotting test was developed (method B). In this method, the liaeniophilic plasnia-agarose layer can be liftcd off when fibrin formation conimenccs so that the position of tlie fibrin formation in tlie haemophilic agarose can be directly compared with thc position
I0
Prudence Bird atrd C.R.Rirzo
of the immunoprecipitatesin the underlying agarose used for the Laurell electroimmunoassay. This has been done for the gel shown in Fig 4(a). The wells in the one-dimensional Laurell agarose, which contained 6% Behringwerke antiserum to factor VIII-related antigen, were filled with dilutions of a normal cryoprecipitate and a haemophilic cryoprecipitate. After electrophoresis the gel was covered with a preformed liaemophilic plasma-agarose gel and when fibrin formation commenced this top gel was removed and both gels were photographed side by side. Fig 4(a) shows the factor VIII-related antigen immunoprecipitates. The faint second line that may be seen beyond the main precipitate (here and in Fig 5 ) results from uneven running of the cryoprecipitate through the agarose and not to a second antibody valency. Fig 4(b) shows the fibrin formation in the upper haemophilic-agarose layer. The heights of the fibrin ‘rockets’ for the normal cryoprecipitate dilutions correspond with that of their immunoprecipitates. Once again the haemophilic cryoprecipitate showed only factor VIII-related antigen and no fibrin formation. To eliminate the possibility that the factor-VIII activity might be simply trapped in the factor VIII-related antigen immunoprecipitate, the one-dimensional Laurell electroimmunoassays were repeated using antiserum in the agarose against plasma proteins other than factor VIII-related antigen. If factor-VIII activity was found to be associated also with these other immunoprecipitates a non-specific trapping mechanism would be suspected. Anti-a, -antitrypsin was chosen for this experiment since a -antitrypsin has a faster electrophoreticmobility than factor-VIII activity, making it necessary for factor-VIII activity to migrate through the a -antitrypsin imniunoprecipitate. One half of the agarose gel shown in Fig 5 contained 7% antiserum to factor VIII-related antigen whilst the other half contained 20% antiserum to a,-antitrypsin. The wells contained cryoprecipitates from normal and haemophilic plasmas and a factor-VIII concentrate. All these samples contained a -antitrypsin. The one-dimensional electroimmunoassay was developed for factor-VIII activity. All the fibrin in the agarose gel containing anti-factor VIIIrelated antigen formed along the immune precipitates whilst in the agarose gel containing anti-a, -antitrypsin, the fibrin formed beyond the immunoprecipitates. This shows that factor-VIII activity migration was not trapped by antie,-antitrypsin immune precipitate. It is therefore unlikely that the anti-factor VIII-related antigen immune precipitate was now specifically trapping factor-VIII activity. It is possible that the rabbit antiserum to factor VIII-related antigen was not monospecific. There may have been one antibody population recognizing and precipitating factor VIIIrelated antigen and another antibody population recognizing factor-VIII activity protein but forming immune complexes which may not be detected by precipitation techniques. To eliminate this possibility, the experiments with the electroimmunoassay were repeated with three different antisera to factor VIII-related antigen in the agarose. The relative quantity of antibodies of different specificities would be expected to vary in the different antisera but in all cases fibrin formation in the superimposed haemophilic plasma-agarose gel coincided with the factor VIII-related antigen immunoprecipitates.
,
DISCUSSION Using an agarose gel overlay containing haemophilic plasma it is now possible to detect
Factor VIII and Rdated Antigen in Agarose
I1
factor-VIII clotting activity directly after it has been electrophoresed into an agarose gel and it is hoped that this test may be adapted to detect factor VIII in other thin layer separating techniques. The specificity of this test for factor-VIII activity has been checked in several ways and whenever possible haemophilic samples from severely affected haemophiliacs have been included as negative controls. This new test is not quantitative for factor VIII although the speed and extent of fibrin formation in the haemophilic plasma agarose seems to be related to the level of factor-VIII activity in the agarose. It was capable of detecting the factor-VIII clotting activity contained in a 15 ~1 sample of plasma and a factor-VIII level of 25% of normal. This thin layer test has allowed us to relate the clotting activity of factor VIII to immunoprecipitates of factor VIII-related antigen. This required a rabbit antibody to factor VIIIrelated antigen which would form precipitates but which did not neutralize factor-VIII activity. The present studies show that all the factor-VIII clotting activity detectable by this method is found in the same place in the agarose gel as the factor VIII-related antigen immunoprecipitates. In these electroimmunoassays,fibrin formation in the haemophilic plasma agarose overlay occurred sooner than when zonal electrophoresis was used, suggesting that factor VIII was concentrated along the immunoprecipitate. This suggests that much smaller quantities of factor-VIII clotting activity may be detected by this method compared to zonal electrophoresis. Control experiments have shown that it is unlikely that factor-VIII activity was being concentrated in this precipitate by a trapping mechanism or that there was an antibody population to factor-VIII activity complexing factor-VIII activity which was distinct to that for factor VIII-related antigen. We are therefore left with the tentative conclusion that the molecule containing factorVIII activity was associated specifically with the factor VIII-related antigen immunoprecipitatc. This suggests that factor-VIII clotting activity and factor VIII-related antigen determinant(s) are present on the same n~olecule. This conclusion is not compatible with the data of Zimmerman & Edgington (1973), who used human antibody to factor VIII attached to beads and found that it removed factor-VIII activity but not factor VIII-related antigen from normal plasma. If, however, only a small percentage of factor VIII-related antigen molecules also express factor-VIII clotting activity this discrepancy could be explained. It is impossible to draw any firm conclusions at this time on the exact relationship of factorVIII activity and factor VIII-related antigen but the work we present here provides evidence to suggest that these activities are very closely related. ACKNOWLEDGMENTS
We should like to thank Dr Rosemary Biggs and Dr D. E. G. Austen for helpful discussion and Mr R. Borrett for his help with the photography. A grant from Action for the Crippled Child is gratefully acknowledged. REFERENCES BIGGS,R. & MACFARLANB, R.G. (1966) Treatment of weight and its aggregation. British Jorrmal .f Haernophilia and Other Coagirlation Disorders, p 3 50. Haerriafology, 27, 89. Blackwell Scientific Publications, Oxford.
AUSTEN, D.E.G. (1974) Factor VIII of small molecular
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Prudence Bird and C . R. Rixza
BIGGS,R. (1972)Human Blood Coagulation, Haemostasis and Thrombosis, p 614.Blackwell Scientific Publications, Oxford. BIRD,P. (1975)Coagulation in an agarose gel and its application to the detection and measurement of factor VIII antibodies. BritishJournal of Haematology, 29s 329. HOYER, L.W. (1973)Specificity of precipitating antibodies in immunological identification of antihaemophilic factor. Nature: New Biology, 245, 49. KERNOFF, P.B.A. (1973)Affinity offactor VIII clotting activity for antigen detectable immunologically. Nature: New Biology, 244, 148. LAURELL, C.-B. (1972) Electroimmunoassay. Scandinavian Journal of Clinical and Laboratory Investigatiorr, 29, SUppl. 124, p 21.
NEWMAN J., JOHNSON,A.J., KARPATKIN, M.H. & PUSZKIN, S . (1971)Methods for the production of clinically effective intermediate and high-purity factor-VIII concentrates. British Joirrnal of Haeniotology, 21, I . RIZZA,C.R. & BIGGS,R. (1973) The treatment of patients who have factor-VIII antibodies. Britisfi Journal of Haetnatology, 24, 65. ZIMMERMAN, T.S. & EDGINGTON, T.S. (1973)Factor VIII coagulant activity and factor VIII-like antigen: independent molecular entities. ]orrrnal of Expcrimental Medicine, 138, 101s. ZIMMERMAN, T.S., RATNOFF, O.D. & POWELL, A.E. (1971) Immunologic differentiation of classic hemophilia (factor VIII deficiency) and von Willebrand’s disease.Journal of Clinical Investigation, 50, 244.
