BrifishJournal offfaematology, 1978, 40, 63 I d 4 I .

Isolation of Human Antibodies to Factor VIII J. M. LAVERGNE, DOMINIQUE MEYER, J. K o m s

AND

MARIE-JOSG LARRIEU

Institut de Pathologie Cellulaire, I N S E R M U 143, Hdpital de BicBtre, Le Kremlin-Bicitre, France (Received

10

February 1978; accepted for publication 4 April 1978)

SUMMARY. It has been claimed that human anti-VII1:C antibodies do not form stable complexes with factor VIII and this fact has hampered in the past the isolation of such antibodies. In this study the purification of human anti-VII1:C antibodies appearing in haemophiliac patients following replacement therapy has been achieved using two different systems. In a liquid phase system, purified human factor VIII was mixed with IgG from a haemophilic patient with a high titre antibody. Specific anti-VII1:C antibodies were recovered following filtration of the antigen-antibody complexes on Biogel A-gm, dissociation of complexes a t pH 3.5 and final isolation by filtration on Sephadex G-zoo. In a solid phase system, the same IgG fraction was specifically bound to insolubilized human factor VIII. Purified anti-VII1:C antibodies were subsequently recovered by elution of antigen-antibody complexes with magnesium chloride. The results demonstrated that stable complexes form between anti-V1II:C antibodies and either the whole factor VIII molecule, or VII1:C dissociated by previous interaction with the antibodies. It is postulated that, in vivo, similar antigen-antibody complexes may form following replacement therapy in haemophilic patients with antibody. Factor VIII/von Willebrand Factor (F.VIII/WF) is a large glycoprotein (M.W. > I x 106 daltons) (Legaz et all 1973) necessary for blood clotting (factor VIII procoagulant activity, V1II:C) and platelet adhesion to subendothelium (von Willebrand factor activity, VIIIR:WF). This protein carries antigenic determinants (factor VIII related antigen, V1IIR:Ag) which react with heterologous antisera prepared against F.VIII/WF. Human anti-factor VIII antibodies which occur in 5-10% of haemophiliacs following replacement therapy are more specific than heterologous antisera (Shapiro, 1975). These antibodies are uniquely directed towards VII1:C and do not neutralize VII1R:WF nor induce precipitation with VII1R:Ag (Shapiro, 1975). The isolation of human anti-V1II:C antibodies has been hampered by the absence of formation of precipitating complexes with factor VIII (Hoyer, 1972). Recently, however, polyethylene glycol has been shown to induce secondary precipitation of immune complexes between factor VIII and anti-VII1:C antibodies (Lavergne et al, 1976). The presence of such complexes has since been confirmed by Lazarchick & Hoyer (1977). In this paper the isolation of human anti-VII1:C antibodies is described using two different Correspondence: Dr Dominique Meyer, I.P.C. (INSERM U 143). H6pital de BicPtre, 94270 le Kremlin-BicCtre, France. 0007-1048/78/1200-063 r$oz.oo

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J . M . Lavergne et a1

systems: liquid phase and solid phase. In the liquid phase system, human F.VIII/WF is mixed with immunoglobulins containing anti-VII1:C antibodies. The mixture is then filtered on agarose, complexes are isolated and anti-VI1I:C antibodies are recovered following dissociation of complexes and filtration on Sephadex G-200. In the solid phase system, anti-VIII:C antibodies are specifically bound to insolubilized F.VIII/WF. Purified anti-VII1:C antibodies are subsequently recovered by elution. Both liquid and solid phase experiments indicate that stable complexes form between anti-VIII:C antibodies and either the whole F.VIII/WF, or VIII:C dissociated from the rest of the molecule by previous interaction with the antibodies. These methods provide in addition a new means for purifying specific homologous anti-V1II:C antibodies. MATERIALS AND METHODS Blood collection. Blood was drawn from normal donors and from a haemophilia A patient with anti-VIII:C antibodies. Nine parts of blood were added to one part of 3.8% w/v trisodium citrate containing 2 mM phenylmethylsulphonylfluoride and 10 u per ml Kunitz inhibitor. Plasma was obtained by centrifugation for 1 5 minat 3000g, 2ooC,and then 3 0 min at Sooog, 4°C. Plasma was either used immediately or frozen in dry ice and stored at - 80°C. A reference pool of 20 normal plasmas, kept at - 80°C for no longer than 2 months, was used as the control in all techniques. Materials. Columns for gel filtration, Sepharose, Sephadex and Dextran Blue 2000 were obtained from Pharmacia Fine Chemicals AB; Biogel A-gm, 200-400 mesh (Bio-Rad) from Touzart et Matignon, Paris; agarose (Indubiose-A 3 7) from Industrie Biologique Franqaise; octanoic acid from British Drug House Chemicals Ltd; imidazole and phenylmethylsulphonylfluoride (P.M.S.F.) from Sigma Chemical Co.; Kunitz inhibitor (Iniprol) from Choay Laboratories, Paris. All other reagents were obtained from Merck. Assay of VZZZ:C and ofanri- VIZZ: C antibodies. VIII:C was estimated using a one-stage method (Langdell et al, 1953) based on the correction of the partial thromboplastin time of severe haemophilia A plasma (V1II:C< I %) in the presence of kaolin and cephalin. Anti-VIII:C activity was measured by reference to residual VII1:C following incubation of equal volumes of standard plasma and either buffer or serially diluted test material for 2 h at 37°C. The titre of anti-VII1:C antibodies was determined as previously described (Lavergne et al, 1976)and expressed in Oxford Units per ml (one inhibitor u per ml inactivates 0.75 u VII1:C per ml). The titre of anti-VI1I:C antibodies was also estimated following dissociation of antigen-antibody complexes by treatment at pH 3.5 for 3 0 min using 0.1M HCI, neutralization a t pH 7.3 using 0.1 M NaOH and centrifugation for 20 min a t 80008, 2ooC,as described by Allain & Frommel (1973). Assay of VZZZR:Ag was performed by the quantitative immunoelectrophoresis technique of Laurel1 (1966) as recently modified (Shoa'i et al, 1977) using rabbit antisera against human F.VIII/WF (Meyer et al, 1972). Purification o f human F. V I I I / WF. Cryoprecipitate obtained from fresh normal plasma was dissolved in imidazole buffered saline (IBS, 0.15 M NaC1, 0.253 M imidazole, pH 7 . 3 ) containing the same protease inhibitors as for the collcction of blood. It was filtered at 20°C on Biogel A-gm ( 5 x 60 cm column) using the same buffer (flow rate 20 ml/h). The first protein

Factor-VIII Antibodies

63 3

peak, corresponding to the void volume (V,,) of the column, was pooled and contained 2-5 u per ml VIII:C and 4-8 u per ml VII1R:Ag. When necessary, such a fraction was concentrated by pressure dialysis against an Amicon PM-30 diaflo membrane (Amicon B.V., Oosterhout, Netherlands). Purified human F.VIII/WF was also prepared by the same method using as starting material factor VIII concentrates kindly supplied by J. P. Soulier, Centre National de Transfusion Sanguine, Paris. This purified human F.VIII/WF was used to raise an antiserum in a goat. This antiserum neutralized V1II:C (titre 40 u/ml) and specifically precipitated VII1R:Ag. Purijcation of immunoglobulins. Immunoglobulins were isolated by the method of Fine & Steinbuch (1970), using 40% ammonium sulphate and octanoic acid. Normal plasma, haemophilia A plasma with anti-VIII:C antibodies (titre 1000 u/ml) and goat anti-human F.VIII/WF antiserum (titre 40 u/ml) were used as starting material. The immunoglobulins were dialysed against 0.15M NaC1, pH 8, and contained approximately 10 mg per ml protein with the original inhibitory properties of starting material. Immunoelectrophoresis was performed on these immunoglobulins in I % agarose, 0.05 M sodium barbital buffer, pH 8.6. One microlitre was electrophoresed at 20°C (60 min, v =3 00 V) and immunoprecipitation was developed with IOO pl of horse anti-human serum antiserum. The immunoelectrophoretic analysis revealed exclusively immunoglobulin IgG. Labelling of immunoglobulins. 20 pg of immunoglobulins from haemophilia A plasma with anti-VII1:C antibodies, referred to as ‘anti-VII1:C immunoglobulins’, were labelled with carrier free 1251 (125-I-S-4, Centre d’Energie Atomique, Saclay, France) by means of the chloramine T method (Hunter & Greenwood, 1962). The labelled ‘anti-VIII:C immunoglobulins’ had a specific activity of 2.5 pCi/pg. Radioactivity was counted in an Intertechnique automatic gamma counter. Isolation of anti- V I I I :C antibodies in liquid phase. In eight separate experiments, human F.VIII/WF, containing 25-50 u VIII:C and 4 0 6 4 u VIIIR:Ag, was incubated with a large excess of unlabelled and labelled ‘anti-VIII:C immunoglobulins’ (400-800 u anti-VIII:C and 8-40 x I O ~[1251]protein-cpm). Following incubation for 2 h at 37°C and 36 h at 4”C, the mixture was filtered on Biogel A-gm as described later. Each peak of absorbance at 280 nm and each peak of radioactivity was pooled and tested for VII1R:Ag and protein. Anti-VI1I:C titre and protein characterization by polyacrylamide gel electrophoresis (see later) were also compared before and after treatment at pH 3.5, Isolation of anti-VIII:C antibodies in d i d phase. In three separate experiments, the immunoglobulins isolated from the goat anti-F.VIII/WF antiserum, which had an anti-VI1I:C titre of 5 u per mg protein, were insolubilized onto Sepharose zB by the cyanogen bromide (CNBr) technique of March et al (1974). The amount of protein coupled was 6-8 mg per ml of Sepharose. The coupled beads were poured into a 0.9 x I S cm column equilibrated with IBS, pH 7.3. The beads were then reacted with an excess of purified human F.VIII/WF until VII1R:Ag and VIII:C were no longer bound to the beads. The beads were finally treated with a mixture of unlabelled and 1251-labelled‘anti-VI1I:C immunoglobulins’. Following extensive washing with IBS and with 0.1M glycine, 0.5 M NaC1, pH 10,elution was performed with 2.5 M magnesium chloride, pH 7.3. Gel jfiltration of immune complexes and free immunoglobulins was performed on Biogel A-sm (1.6 x IOO cm column), using IBS, pH 7.3, at 20°C with a flow rate of 10 ml/h. Elution

f. M . Lavergne et a1

634

was maintained using a peristaltic pump. Dextran Blue 2000 and purified IgG were used as markers. The peaks obtained by gel filtration on Biogel A-gm were passed through a 1.5 x 90 cm column of Sephadex G 200, before and after treatment at pH 3.5. The buffers used were either IBS pH 7.3 or 0.05 M glycine, 0 . 1 M NaCl, pH 3 . 5 . The flow rate was 3 . 5 ml/h. Protein concentration was measured by the method of Lowry et a1 (195 I ) . Polyacrylamide gel electrophoresis was performed according to Davis & Ornstein (Sargent, 1969) in 4% gels, 0.05 M Tris, 0 . 3 8 M glycine buffer, pH 8.3 (5 mA per gel, 40 min). Gels were either stained or frozen in dry ice and sliced in order to count the radioactivity associated with each 2 mm segment. RESULTS Isolation of Immune Complexes in Liquid Phase Eight experiments were performed with mixtures of purified F.VIII/WF (25-50 u VII1:C) and large excess ( x 15-30) of 1251-labelled'anti-VII1:C immunoglobulins' (400-800 u antiVII1:C). Following incubation a t 4'C, the mixture contained VI1IR:Ag and anti-V1II:C activity, but no detectable VI1I:C procoagulant activity. This mixture was filtered on Biogel A-gm and three peaks of absorbance at 280 nm which corresponded to three peaks of radioactivity were consistently observed (Fig I). The first peak, corresponding to the V , of the column, contained most of the VII1R:Ag (>98% of the total amount recovered) and between 0.4 and 1.5% of radioactivity. No anti-VI1I:C was found but some was recovered following treatment at pH 3 . 5 (between 3.4 and 9.8 u, representing 0.7-1.6% of the starting material (Table I). The third peak, corresponding to the elution position of the bulk of IgG, contained no VIIIR:Ag, but most of the anti-VIII:C (54-70%) and radioactivity (74-93% of the starting material). Following treatment at pH 3.5, the amount of anti-V1II:C did not increase in this fraction. The ratio ofanti-VI1I:C activity to [1251jprotein(0.35 x I O - ~ ) was only slightly lower Absorbance ( 2 8 0 mn)

vo

------

1

I protein (cpm x lo3)

n

.lo 000

1

Vlll R :AG CU/ml) 200.

ISG

i-f

-.-.-.-

ii ,150

100.

,100 I

50

. o

0. 60

120

180

Volume Cml) FIG I . gel filtration on Biogel A-gm of a mixture of purified F.VIII/WF ( 3 0 u VII1:C and 42 u VI1IR:Ag) and 1251 'anti-V1II:C immunoglobulins' (550 u anti-V1II:C).

Factor- VZIZ Antibodies

63 5

TABLE I. Isolation of anti-VI1I:C antibodies in liquid phase: results from eight experiments

VII1R:Ag Units*

%t

[ 1251]protein cpm x 103*

%t Anti-VIIIC, before pH 3.5 units*

%t

Anti-VIII:C, after pH 3 . 5 units*

%t

Proteins mg*

%t

39 (27-41) 63-8 I

0.7 (0.0-1.1)

124 (67-204) 0.4-1.5

478 (151-1160)

-

0

5.8 (4.4-7.3) 0.7-1.5

327.2 (245-409) 5449.8

6.6 (3.4-9.8) 0.7-1.6

74.6 (53.4-95.7) 9.1-21

336 (262-400) 58-68

1.9 (1.5-2.2)

1.4 (1.3-1.5) 15.4-21.4

25.5-34.6

0-1

1.9-3

0

14520 ( 4 0 0 ~ 3 1 5 0 0 ) 74-93

2.4 (2.-2.9) 3 0.7-46

* Total recovered (mean and extreme values).

t Percentage of applied material on the column (extreme values). than that of the starting material (0.40 x I O - ~ ) . The second peak contained traces of VI1IR:Ag with 0.7-1.5 % anti-VI1I:C activity and 1.9-3 % of applied [ 1251]protein. Anti-VII1:C increased 10-30-fold following treatment at pH 3.5. The ratio of anti-VII1:C activity to ) a 15-2s-fold relative purification as compared with the [1251]protein(0.33 x I O - ~ suggested starting material. Polyacrylamide gel electrophoresis was performed on each of the three peaks. The third peak showed no change before or after treatment at pH 3.5, the radioactivity being found in a position corresponding to that of purified IgG (Fig 2). Most of the radioactivity of the second peak which was found near the origin before pH 3.5 shifted to the IgG position after pH 3.5. The same data were observed for the first peak. Sephadex G-zoo filtration was also performed on each of the three peaks, at both pH 7.3 and 3.5. Similar to results by polyacrylamide gel electrophoresis, the third peak eluted in the IgG position at both pH 7.3 and 3.5. In the first and second peaks, there was a shift of the [1251]proteinto the IgG position following treatment at pH 3.5 (Fig 3).

Isolation oflmmune Complexes in Solid Phase The principle of the method used is shown in Fig 4. In three experiments, goat antiF.VIII/WF IgG was coupled to cyanogen bromide-activated Sepharose. An excess of purified human F.VIII/WF was circulated through the column until VII1R:Ag and VII1:C were no longer bound to the beads. 425680 u V1II:C was estimated to be bound. The 1251-labelled ‘anti-VII1:C immunoglobulins’ which were finally reacted with the beads contained 500-1 100 u anti-VIIIC, and 17-28 x 106 [1251]protein-cpm. Following extensive washing in order to

J . M . Lavergne et a1 1" I protein ccpm x 1 0 9

1

r

0 2 4

I

0 0 1012 1416102022242028

Distmce from origin Cmm>

FIG 2 . Polyacrylamide gel electrophoresis of the three peaks obtained following gel filtration on Biogel A-gm: , beforepH3.5;-----, after pH 3.5. l n 5 1 protein (cpm x W )

1

60

Volume C ml 1 FIG 3 . Gel filtration on Sephadex G-zoo of the second peak obtained following gel filtration on , before pH 3.5; -----, after pH 3.5. Biogel A-gm: -

Factor- VZZZ Antibodies

63 7

FIG4.Isolation of anti-VI1I:C antibodies in solid phase. Principle of the experimental procedure. IgG from goat anti-F.VIII/WF antiserum were insolubilized onto Sepharose beads. F.VIII/WF was then reacted with the IgG beads. Finally the beads were treated with 1251-labelled‘anti-VIII:C IgG’. The arrows point to the theoretical sites of action of magnesium chloride.

Absorbance ( 280 mn)

1151 protein (cpm x lo3)

------

- 30 0.050

-

0

60

120

180

Volume ( m i ) FIG 5. Gel filtration on Biogel A-Sm of the MgClz elution peak.

remove non-specifically adsorbed material, between 77 and 79% of the applied anti-VIII:C inhibitory activity, with 8 I-92% of radioactivity, were recovered in the combined effluents. After elution with 2.5 M magnesium chloride, one peak was obtained which contained 0.32-0.37% of the original [1251]protein. This elution peak was dialysed against 0.25% ethylenediaminetetraacetic acid and filtered on Biogel A-gm using IBS, pH 7.3. Three peaks of radioactivity with corresponding absorbance at 280 nm were obtained (Fig 5 ) . From 8 to I I % of radioactivity found in the MgC12 eluate was recovered in the first peak (Table 11), 32-77% in the second and 12-60% in the third, corresponding to the elution position of purified IgG. Only peaks I and I1 contained anti-VIIK complexes dissociableat pH 3.5 (Table 11). In peak 111, anti-VIII:C activity did not increase following treatment at pH 3.5. The ratio of anti-VII1:C activity to [1251]protein in the second peak following treatment at pH 3.5 ( 0 . 3 6 ~10-l) suggests a Ioo-fold relative purification as compared to the starting material (0.32 x I O - ~ ) .

J . M . Lavergne et a1 TABLE 11. Isolation of anti-VIII:C antibodies in solid phase: results from three experiments

[1251]protein cpm x 103 ?hof starting material* % of material eluted after MgClzt Anti-VIII:C, before pH 3 . 5 units % of starting material Anti-VIII:C, after pH 3 . 5 units YOof starting material

4.5-11.8 0.02+.04 8.2-1 1.2

17.8-80.7 0.1-0.28 3I4~76.8

12.6-33.3 0.04-0.19 12-59.8

0.6-2

67.5-1 16 6.1-10.5

2*43 1.8-8.5

6.1-37 I .2-3.3

94-164 14.8-1 8.7

I .7-8.9

3.1-23

1945

* Ratio of recovered [1251]proteinat the final step (gel filtration on Biogel A-Sm) to total

amount applied on the immuno-adsorption column.

t Ratio of recovered [ 1251]proteinat the final step to amount recoveredfollowing

elution with MgC12.

DISCUSSION Heterologous antisera directed towards F.VIII/WF (VII1R:Ag-V1II:C) lead to the formation of precipitating complexes with the molecule (Zimmerman et al, 1971; Meyer ef al, 1972).O n the contrary it has usually been reported that human anti-VIII:C antibodies do not form stable complexes with F.VIII/WF (Hoyer, 1972). Recently, however, specific immune complexes have been isolated by precipitation by polyethylene glycol (Lavergne et al, 1976).These results have led us to reinvestigate the binding properties ofhuman antibodies to VIII:C, isolated from patients with haemophilia A. This has been achieved by two procedures: in liquid and solid phase. Our data indicate that complexes in fact form between anti-VII1:C antibodies and F.VIII/WF or VIII:C alone. Such complexes may then be dissociated to some extent by lowering the pH as demonstrated by Allain & Frommel (1973). Anti-VII1:C antibodies are stable at pH 3 . 5 and the loss of antigenic reactivity a t that pH should prevent secondary reassociation (Lazarchick & Hoyer, 1977). Agarose gel filtration allows the separation of complexes and proteins with a large molecular weight (> 106 daltons) from smaller molecules. This property has been used to demonstrate the formation of complexes of anti-VIII:C antibodies with an antigen that has a molecular weight larger than IgG, but smaller than F.VIII/WF. Bound and free IgG may therefore effectively be separated by agarose gel filtration. Complexed anti-VIII:C antibodies were detected in the void volume as well as in the column fractions eluted later but prior to the IgG fraction. In the void volume fractions, no anti-VIII:C activity was demonstrated before treatment at pH 3.5 but only following dissociation of complexes. In the late eluting complexes with a molecular weight greater than that of IgG, anti-V1II:C activity was present before dissociation and increased up to 12-fold following treatment at low pH. The demonstration of anti-VIII:C activity in some fractions prior to dissociation of the antigen-antibody complexes indicates that bound but active antibody was present in excess. There were only traces of

Factor- Vl11 Antibodies

639

VIIIR:Ag, suggesting that the majority of the complexes were formed between anti-VIII:C antibodies and a smaller molecule, possibly VII1:C. The dissociation in the void volume and later fractions has been confirmed by polyacrylamide gel electrophoresis. No complexes were present in the last peak corresponding to the IgG since treatment a t low pH did not result in any increase of anti-VIII:C activity. These results demonstrate that anti-VII1:C antibodies form stable complexes, not dissociated by gel filtration. It suggests that these antigen-antibody complexes involve either the whole F.VIII/WF molecule (VII1R:Ag-VII1:C) or VII1:C alone. The separation of VIII:C from the rest of the F.VIII/WF molecule is unlikely to be the result of the activation of a protease since protease inhibitors were present throughout the study. O n the contrary, it has previously been shown that F.VIII/WF can be separated into two distinct moieties (WF and VII1:C) by interaction with insolubilized homologous anti-VII1:C antibodies (Zimmerman & Edgington, 1973; Koutts et al, 1977) and our data are in agreement with those results. Recently Lazarchick & Hoyer (1977) have also demonstrated the presence of complexed anti-V1II:C antibodies. When plasma containing anti-VIII:C antibodies was mixed with cryoprecipitate, specific immune complexes were detected in similar gel filtration fractions to those in our experiments. These authors showed that even in antibody excess, only a small amount of large immune complexes was formed, and that most complexes were of smaller molecular weight. It seems unlikely, however, as suggested by Lazarchick & Hoyer (1977)~that the VII1:C part of the F.VIII/WF molecule is monovalent. In preliminary data, we have found that VIII:C bound to insolubilized human anti-VI1I:C antibodies (Koutts et al, 1977) is still capable of neutralizing an additional source of anti-VIII:C antibodies. In our study, the greater yield of anti-VII1:C antibodies (as compared to that of Lazarchick & Hoyer, 1977) may be related to the higher titre of the antibody used, the longer time of incubation of the antigen-antibody mixture and the large excess of antibody in our experimental conditions. The isolation of specific anti-VIII:C antibodies has been achieved in the liquid phase system following dissociation of complexes at low pH and final separation by filtration on Sephadex G-200. The other procedure, employing immobilized reactants, provides an independent means of isolating these antibodies. The binding of F.VIII/WF to heterologous anti-F.VIII/WF IgG coupled to activated Sepharose beads had previously been demonstrated by Zimmerman & Edgington (I973), Peake & Bloom (1976) and Koutts et al (1976). Such F.VIII/WF beads have then been used to specifically bind anti-VIII:C antibodies, and separate them from the bulk of immunoglobulins. Specific anti-VII1:C antibodies were finally eluted with magnesium chloride and submitted to gel filtration on agarose. The pattern of elution was similar to that of the liquid phase system. This indicates that magnesium chloride dissociates antigen-antibody complexes at various sites: between goat anti-F.VIII/WF IgG and F.VIII/WF as well as between F.VIII/WF and anti-VIII:C antibodies (Fig 4). In addition, magnesium chloride must have dissociated F.VIII/WF into two moieties (VII1R:Ag and VIILC), like high salt (Rick & Hoyer, 1973; Meyer et al, 1974) or calcium chloride (Owen & Wagner, 1972). It is thus conceivable that complexes between VII1:C and anti-VI1I:C antibodies are also obtained using this solid-phase procedure. Anti-VI1I:C activity increased following dissociation of antigenantibody complexes at low pH, although to a lesser extent than with the liquid phase system. This difference, however, is probably related to the totally distinct experimental conditions which do not allow a direct comparison of the results.

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J . M . Lavergne et

a1

The solid phase procedure, although cumbersome, has the advantage of yielding more purified anti-VIII:C antibodies. The third peak in particular is composed only of purified anti-VII1:C antibodies, separated from the bulk of immunoglobulins by previous immunoadsorption. In the liquid phase system, the majority of these antibodies are present in the third peak, together with the bulk of IgG, and only the first fractions yield purified antibodies following dissociation and gel filtration on Sephadex G-200. Results using the solid phase system indicate that in the haemophilic patient studied, the amount of anti-VIII:C antibody represents less than 2% of the total IgG fraction. In conclusion, stable complexes form between VIII:C and anti-VI1I:C antibodies. The purified antibodies obtained following dissociation and Sephadex G-200 filtration should be useful for immunochemical studies, such as characterization of heavy and light chains, or development of an immunoradiometric assay for the VII1:C component of the F.VIII/WF molecule(s). It is tempting to postulate that the formation of complexes between VIII:C and anti-VIII:C antibodies may also occur in vivo in haemophilic patients (with an antibody) following replacement therapy. The formation of such complexes would lead to the dissociation of the F.VIII/WF molecule. There might be in such cases two types of complexes with a different clearance: large complexes between the whole F.VIII/WF and anti-VI1I:C antibodies, and smaller complexes involving only V1II:C.

ACKNOWLEDGMENTS

W e would like to thank N. Ardaillou and J. P. Girma for labelling of immunoglobulins with 1251 and T. S. Edgington (Scripps Clinic and Research Foundation, La Jolla, Ca, U.S.A.) for helpful comments and suggestions during this investigation. The technical help of B. Obert, C. Mezerette, C . Jacobson and N. Thomas is gratefully acknowledged. REFERENCES

D. (1973) Antibodies to ALLAIN, J.P. & FROMMEL, Factor VIII. I. Variations in stability of antigenantibody complexes in hemophilia A. Blood, 42, 43 7-444.

FINE,J.M. & STEINBUCH, M. (1970) A simple technique for the isolation of monoclonal IgG and IgA. Revue EuropPenne &Etudes Cliniques et Biologiques, 15, 1 1 1 5 - 1 1 2 1 . HOYER, L.W. (1972) Immunologic studies of antihemophilic factor (AHF, factor VIII). 111. Comparative binding properties of human and rabbit antiAHF. Blood, 39,481-489. HUNTER, W.M. 81 GREENWOOD, F.C. (1962)Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature, 194, 495-496.

K o m s , J., GUDE,N. & FIRKIN,B.G. (1976) The dynamic inter-relationship between factor VIII and von Willebrand factor. Thrombosis Research, 8. 5 3 3-5 41.

K o m s , J., LAVERCNE, J.M. & MEYER, D. (1977)Immunological evidence that human factor VIII is composed of two linked moieties. BritishJoournal of Haematology, 37, 415-428. LANCDELL, R., WAGNER, R.H. & BRINKHOUS, K.M. (1953) Effect of antihemophilic factor on one-stage clotting tests.journal ofLaboratory and Clinical Medicine, 41,637-647 LAURELL, C.B. (1966)Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analytical Biochemistry, 15, 45-52. LAVERGENE, J.M., MEYER,D. & REISNER, H. (1976) Characterization of human anti-factor VIII antibodies purified by immune complex formation. Blood, 48,93 1-939. J. & HOYER, L.W. (1977) The properties LAZARCHICK, ofimmune complexes formed by human antibodies to factor VIII. journal of Clinical Investigation, 60, IO7eI079.

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Factor- VIZI Antibodies E.W. (1973) Isolation and characterization of human factor VIII (antihemophilic factor). Journal of Biological Chemistry, 248, 3946-3955. LOWRY, O.H., ROSEBROUGH, N.J., FARR,A.L. & RANDALL,RJ. (1951) Protein measurement with the folin phenol reagent.Journa1 ofBiologica1 Chemistry, 193, a65-275.

MARCH,S.C., PARIKH, I. & CUATRECASAS, P. (1974)A simplified method for cyanogen bromide activation of agarose affinity chromatography. Analytical Biochemistry, 60,149-152. MEYER,D., JENKINS, C.S.P., DREYFUS, M.D., FRESM.-J. (1974) Willebrand SINAUD, E. & LARRIEU, factor and ristocetin. 11. Relationship between Willebrand factor, Willebrand antigen and factor-VIII activity. British Journalof Haematology, 28,579-599. MEYER, D., LAVERGNE, J.M., LARRIEU, M.J. &Josso, F. (1972) Cross-reacting material in congenital factor VIII deficiencies (haemophilia A and von Willebrand’s disease). Thrombosis Research, I, 183-196.

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