Transfusion Medicine, 1992, 2,223-229

Development, optimization and use of an enzyme linked immunosorbent assay (ELISA) to measure factor VIII antigen utilizing monoclonal antibodies V. S. Hornsey, 0. Drummond, P. Eaglesfield,* D. S. Pepper and C. V. Prowse Scottirh National Blood Transfusion Service, Notional Science Luborotory. Edinburgh and *SNBTS. Protein Fractionation Centre. Edinburgh. Scotland Received I.? September 1991; accepted for publication I.? December 1991

SUMMARY.An enzyme linked immunosorbent assay

Forty-eight samples with VII1:Ag levels ranging from 0.006 to 1.5 u/ml were assayed by both ELISA and IRMA. The coefficient of correlation between the two assays was 0.89. In addition to measuring human VIII:Ag, it is also possible to detect antigen in several animal plasma and sera.

(ELISA) has been developed to measure VI1I:Ag in plasma and concentrates. The assay utilizes two commercially available monoclonal antibodies to VII1:Ag and provides an alternative to the established immunoradiometric assay (IRMA). It has the advantage of not requiring the use of radioactive material and human antibodies. The assay sensitivity is 0.006 u/ ml and the interassay coefficient of variation is 6.3%.

Key words VIII:Ag, ELISA, monoclonal antibodies.

Tuddenham (198 I), as described by Griffin (1986), and a two-site solid phase assay (Hornsey et al., 1987). Due to the unavailability, until recently, of appropriate immunogens and the relatively poor immunogenicity of human VIII in laboratory animals, IRMA methods have usually made use of human antibodies. These are in limited supply, especially those of high titre which are the most useful. Furthermore, reagents prepared from human plasma may be infective due to viral contamination and are therefore best avoided. Following the development of monoclonal antibodies (mAb) to VII1:Ag (Muller et al., 1981; Rotblat et al., 1983; Fulcher et al., 1985; Goodall & Meyer, 1985; Griffin et al., 1986), Stel et al. (1983) developed a one-step IRMA using mAb. However, ELISA are increasingly being used in place of radioimmunoassays as they do not require radioactive material and the reagents may be stored for many months. ELISA for VIII:Ag, which use human antibodies, have been described by Dinesen & Feddersen (1983), Nordfang ef al. (1985) and Ingerslev & Stenberg (1986). Hellstern et al. (1987) developed an assay using a mixture of human antibodies and mAb. One ELISA using only mAb has been described by Furlong et al. (1988) and has been used in the identification of haemophilia carriers.

While the activity of factor VIII (VII1:C) is labile, the antigenic determinant (VII1:Ag) is stable in plasma at 37°C for at least 24 h (Peake et al., 1979). The assay of VII1:Ag has allowed improved diagnosis of haemophilia A (Peake et al., 1981) and has proved useful in examining the behaviour of VII1:Ag after transfusion of factor VIII concentrates into haemophiliacs. It is also useful when monitoring factor VIII purification processes. While VII1:Ag may be detected by the consumption of inhibitors in a clotting assay, Peake er at. (1979) and Lazarchick & Hoyer (1978) developed practical immunoradiometric assays (IRMA) using human antibodies which arise in some multi-transfused haemophiliacs. Acquired inhibitors of VIII may also be used. Various types of IRMA, including one-site fluid phase (Lazarchick & Hoyer, 1978; Reisner er al., 1979) and two-site solid phase (Holmberg et al., 1979; Peake et al., 1979) are now in use. Burke et al. (1986) have developed a radioimmunoassay for VIII heavy-chain which uses a canine inhibitor antibody and 1251-VIIIheavy chain. The methods used in this laboratory are a one-site fluid phase IRMA based on the method of Rotblat & Correspondence: V. S. Hornsey, SNBTS, National Science Laboratory. 2 Forrest Road, Edinburgh, EHI 2QN. Scotland,

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224 V. S. Hornsey et a]. The primary aim of this study was to develop an ELISA for VII1:Ag utilizing the panel of mAb (now commercially available) characterized by Griffin et al. (1986). The developed ELISA, which would be compared with the IRMA methods currently used in this laboratory, was to be sensitive and to operate in the range from 0.01 (as in severe haemophilic plasma) to 2-0 (normal plasma = 0.5-2.0) u/ml VII1:Ag. Secondly, the assay was to be used to measure antigen in cryosupernatant plasma, and in samples taken at various stages of factor VIII concentrate production. Thirdly, the assay should be rapid, robust and reliable, allowing use in both routine and research environments. MATERIALS A N D M E T H O D S Reagents

Sephacryl S-200 and Sephacryl S-1000 were obtained from Pharmacia (U.K.) Ltd, Horseradish peroxidase (HRPO) Type VI and bovine serum albumin without preservative (BSA) were from Sigma. One-gram tablets of urea hydrogen peroxide and 2,4-dinitro-lfluorobenzene (FDNB) were from BDH, and 3,3',5,5'tetramethylbenzidine (TMB) GOLD LABEL was from Aldrich. British plasma standards for factor VIIIrelated activities (provided by the National Institute for Biological Standards and Control) were used to develop the assay of plasma samples. Plasma samples for assay were obtained from standard citrate phosphate dextrose adenine (CPD-A 1) blood donations. Factor VIII concentrates and samples from various stages of VIII concentrate production were from the Scottish National Blood Transfusion Service Protein Fractionation Centre (PFC). Sterile pyrogen-free distilled water and isotonic saline were also from PFC. All other reagents were of analytical grade. Selected mAb are commercially available from Bioscot Ltd, Fleming Road, Kirkton Campus for Science and Techklogy, Livingston EH54 7BN, U.K. or from American Diagnostica Inc., 222 Railroad Avenue, P.O. Box 1165, Greenwich CT 06836-1165, U.S.A. Statistical methods The coefficient of variation was determined by repeated assay of distinct dilutions of replicate samples of frozen plasma (stored at -40°C). Correlation between results of the IRMA and ELISA methods was determined from the Pearson parametric coefficient and by linear regression analysis. Protein preparations

Monoclonal antibodies to VI1I:Ag ESH 1-10 were

prepared and purified on protein A coupled to either Sepharose 4B or Sephacryl S-1000 (Griffin et al., 1986). Conjugates of IgG and horseradish peroxidase were prepared by the method of Nakane & Kawaoi ( I 974) and stored in aliquots at -40°C. Under these conditions they were stable for at least 6 months. Assays

v W f A g was measured by RIA (Hornsey, 1988). IRMA for VII1:Ag. The one-site fluid phase and two-site solid phase IRMA have previously been described (Griffin, 1986; Hornsey et al., 1987). ELISA. Optimal conditions will be described here with variations being described in the Results section. Reagents Coating buffer. NaHCO, (50 mM) pH 9.6 or 15 mM phosphate, 150 mM NaCl 7.4 (both gave identical results). Wash buffer. Phosphate (15 mM), 150 mM NaCl, 0.05Y0Tween 20, pH 7.4. Sample buffer. Wash buffer plus 0.3% w/v BSA. Conjugate buffer. Sample buffer plus 0.5 M NaCl. TMB. 10 mg/ml in dimethyl sulphoxide (freshly prepared). Urea hydrogen peroxide. One-gram tablet (33-35% peroxide) dissolved in 100 ml HzO (freshly prepared). Acetate citrate buffer. Sodium acetate (0.1 M), pH adjusted to 6.0 using I M citric acid. Substrate solution. This is based on the method of Bos et af. (1981). Immediately prior to use, 0.2 ml of TMB solution (final 100 pg/ml) and 0.3 ml urea hydrogen peroxide (final 50 pg/ml) were added to 20 ml acetate citrate buffer. ELISA method

Wells of the Titertek Immuno Assay-Plate (high activated), 96 well PVC microtrays (flat-bottomed), cat. no. 77-172-05, were precoated with 200 p1 of mAb diluted to 10 pg/ml in coating buffer. The trays were covered and incubated overnight at 20°C. They were then washed (four times) with wash buffer (200 pl/well) and any remaining sites were blocked for 1 h at 20°C with 200 p1 of wash buffer containing 1% BSA. The trays were again washed and drained by inverting on absorbent paper. One hundred microlitres per well of standard or test, diluted in sample buffer, were added with 100 pl of mAb conjugated to horseradish peroxidase (diluted in conjugate buffer). The trays were covered and incubated a t 20°C for 2 h. Column 1 was filled with 200 pI buffer only. The plasma standard was

ELISA for VIII:Ag 225

diluted 1/5, 1/10, 1/20, 1/40, 1/80 and 1/160. Test samples were diluted to read within the 1/10-1/40 range. Concentrate standards were prediluted to 1 u/ ml of VII1:C and then diluted as above. The trays were again washed ( x 4) and drained following incubation. Two hundred microlitres substrate were added to each well and the reaction was stopped with 50 p13 M HS04 after 15 min. Absorbance was read at 450 nm using a Dynatech MR 5000 plate reader blanked on column 1. Results were interpolated from a plot of the log standard concentration versus the log absorbance.

excess antibody (Hornsey el al., 1988). It was therefore decided to use this as coating antibody. ESHCHRPO was prepared and in the assay gave an OD 450 nm of greater than 1.0 for a 1/5 dilution of plasma standard. Other combinations of mAb pairs from the available panel yielded inferior results in terms of colour yield or assay consistency. Studies by other groups have shown that ESH4 and ESH8 are directed towards the light chain of factor VIII (M. Ezban & D. Scandella, personal communications). Assay optimization

Gel filtration

Normal plasma, serum, haemophilic plasma with a normal V1II:Ag level, high purity VIII concentrate and VIII concentrate treated with thrombin were gel filtered on a Superose 6 column (HR 10/30) on a Pharmacia FPLC system at 0.5 ml/min in 20 mM Tris/ HCl, 20 mM citric acid, 0.1 M NaCl pH 7.4 at 20°C. One millilitre fractions were collected and assayed for VIII:C, VII1:Ag and vWf:Ag. The molecular weight calibration was performed using standards from Pharmacia.

ELISA: assay reproducibiiiiy and correlation with IRMA

RESULTS Preliminary selection of antibody pairs

Immobilized ESH8 from the available mAb panel, has previously been shown to exhibit the highest binding of plasma VIII, removing 88% V1II:Ag and 68% VI1I:C from plasma and cryoprecipitate under conditions of

Fig. 1. Correlation of ELISA with IRMA for plasmas (O), haemophilic plasmas (v),cryosupernatants (0) and plasmas from patients with von Willebrand's disease (A).

Various concentrations of TMB, hydrogen peroxide, ESH8 and NaCl were tried and the best results were obtained using the conditions described in the Materials and Methods section. Sequential incubation with a sample followed by conjugate was also shown to be of no benefit. Assays on haemophilic plasmas yielded very low or blank levels of absorbance. A linear doseresponse curve was obtained with absorbance ranging from 0.2 to 1.0 for plasma standard dilutions of 1/160 to 1/5.

A range of plasmas was assayed by both ELISA and IRMA. Some haemophilic plasmas gave values of less than 0.006 u/ml and cryosupernatant plasma gave a parallel dose-response curve to that of the standard. When a normal plasma was assayed repeatedly against the first British standard 85/644 (VIII:Ag= 1.06 u/ml),

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a potency of 1.05 k0.04u/ml was obtained (interassay coefficient of variation 4.0%, n= 7). The coefficient of variation for the one-site IRMA was 23%. Intra-assay variation was 2.3% (n = 9). Forty-eight samples, including 27 normal plasmas, eight haemophilic plasmas, 11 plasmas from von Willebrand patients and two cryosupernatant plasmas, were assayed by both ELISA and IRMA. The correlation coefficient was 0.89 (Fig. 1). Nine serum samples were assayed by ELISA and the results were not significantly different from the corresponding plasmas with serum V1II:Ag levels being 1.24k0.34 u/ml and plasma levels being 1-15 f0-27. Conjugate which had been stored for 6 months at - 40°C produced a similar colour yield, with standard plasma, to that obtained when the conjugate was freshly prepared.

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Assay of concentrates

Eighteen samples of factor VIII concentrates and samples taken during VIII concentrate production were assayed by both ELISA and IRMA in pilot studies. Values ranged from 10 to 20 u/ml and the correlation between assays was 0.95: using a plasma standard. However, over a 2-year period, non-parallelism between plasma standards and concentrates has frequently been noted, possibly due to the varying nature of concentrate samples obtained from the purification process. It is therefore felt preferable to use a plasma standard for plasma samples and a concentrate standard for concentrate samples. This approach has consistently yielded parallelism between standard and test sample curves (Fig. 2). More recently, 37 concentrate samples (in-process and final product) have been tested in the ELISA system and have all been parallel with the concentrate standard. Gel filtration

VIII:C, VII1:Ag and vWf:Ag for normal plasma were all eluted in the void volume with a small percentage of the VII1:Ag eluting in the protein peak of apparent M, 30,000. In serum, the vWfAg was again in the void volume together with a very small amount of VII1:Ag. The main peak of VII1:Ag eluted at M , 30,000. These results were seen more clearly when VIII concentrate was gel filtered. Preincubation with thrombin again shifted the VII1:Ag peak from the void volume to an M , of 30,000. The CRM+ haemophilic plasma showed the VII1:Ag peak to elute with an M , of between 300,000 and 400,000 (Fig. 3).

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Fig. 2. Dilution curves for the first British plasma standard 85/644 (a), normal plasma (A), VIII concentrate dilution 1 u/ml (O),eighth British concentrate standard 87/658 diluted to 1 u/ml (A) and haemophilic plasma (0). VI1I:Ag concentration of the concentrate standard was assumed to be the same as that of VII1:C.

DISCUSSION This study describes the development of an ELISA for measuring VI1I:Ag in plasma and concentrates. It uses two mAb to VII1:Ag and provides an alternative to the established IRMA with the advantage of not requiring radioactive materials and human antibodies. This ELISA is simpler to perform than the IRMA, gives consistent results and the reagents are stable at -40°C for at least 6 months. It uses whole IgG and therefore no Fab' preparation is required. In assays of this type, the use of mAb is advantageous as they can be produced in practically unlimited quantities giving reagents which do not vary. As each mAb is directed to a different specific epitope, the sample may be incubated with the coating and conjugated antibodies simultaneously, thus reducing the assay time. It might

ELISA for VIII:Ag 227

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Fig. 3. Gel filtration on Superose 6 of (a) plasma, (b) serum, (c) haemophilic plasma, (d) VIII concentrate and (e) VIII concentrate treated with thrombin. VI11:Ag (-) vWf:Ag (.-.-. ) VI1I:C ( * * * ).

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be considered desirable, but not essential, to use more than one coating antibody in case one of them recognizes an epitope not universally present in all plasma-derived samples, i.e. a polymorphic epitope (Stel et af., 1983). However, this has not, in our experience, been a problem. TMB was chosen as chromogen as it has been shown by Bos et af. (1981) to be highly sensitive for HRPO in ELISA. Unlike benzidine it is not carcinogenic. Only a few serum samples were tested in this ELISA and the results were not significantly different from the corresponding plasma samples. This contrasts with the results of the IRMA of Holmberg et af. (1979), who found lower levels in serum, but agrees with Peake et af. (1979). Hada et af. (1987), using ESH8 as a second antibody in an ELISA with human coating antibodies, found no difference between serum and plasma VII1:Ag levels but they also describe another antibody which failed to recognize serum VII1:Ag. These discrepancies are presumably due to differences in the specificities of the antibodies used. However, these specificities can be well defined when using mAb reagents. The ELISA developed by Nordfang et af. (1985), and the IRMA developed by Stel et af. (1983), have sensitivities of 0.002 and 0.0005 u/ml respectively. The

ELISA developed here has a sensitivity of 0-006u/ml and compares well with that of the IRMA methods used in this laboratory (0.005 u/ml). The coefficient of variation for the local one-site IRMA was 23% and this greater variability of the IRMA may account for the few discordant results seen when comparing the two assays. Both EDTA and NaCl are known to dissociate VIII from vWf (Tran & Duckert, 1983) thereby potentially exposing epitopes on VII1:Ag not seen when it is complexed to vWf. EDTA also dissociates the heavy and light chains of VII1:C (Fass et al., 1982) thus causing the dissociation of metal ion bridged heterodimers or heterotrimers, again possibly exposing otherwise hidden epitopes. Hada et af. (1987) showed that when they used ESH8 as a second antibody in their ELISA, adding 0.2 u/ml thrombin to plasma increased the apparent VII1:Ag content, although this was not observed in serum. In this work, coating with ESH4 and the use of ESH8-HRPO as a conjugate were also investigated. However, exceptionally high results were obtained with some plasmas. It is possible that this combination is affected by proteolysis of VIII in these samples as this assay format was also shown to be affected by the addition of EDTA and NaCl. Coating with ESH8 and

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using ESHCHRPO was much less sensitive to such variations, giving a more robust assay. In particular, it was insensitive to variations in NaCl concentration. Why concentrate samples should not consistently yield dilution curves parallel to those for a plasma standard in this assay system is not known. Changes in batches of antibody and slight variations in the method of conjugation have occurred and it is possible that the assay is not robust enough to be unaffected by these. The type of standards and concentrates have also changed over the past 2 years. Whatever the reason it is a problem, particularly as there is, as yet, no distinct concentrate standard available. One of the aims of this work was to be able to assess samples from VIII concentrate production and determine whether the losses in VII1:C during processing were due to inactivation or were mechanical. In this laboratory, the problem has been addressed by analysing results in terms of the ratio of VII1:Ag to activity (VIII:C), relative to that assayed in a standard concentrate. For example, the V1II:c to VII1:Ag ratio of the intermediate purity VIII concentrate was 1.8 fO.4 ( n = 6 ) . After additional chromatography to yield a high potency factor VIII, this ratio was 1.6 f0-4. Thus the ratios have remained relatively constant implying that the VIII molecule has not been greatly altered during this stage of the process and that any losses are largely mechanical. These relative ratios give an excellent guide to what is happening during production. However, an appropriate concentrate standard would be needed to be able to derive absolute potencies. Ultimately this problem could be solved by the availability of pure recombinant VIII of known concentration and activity. Gel filtration showed that VII1:Ag is associated with both active and inactive protein in plasma. In both serum and VIII concentrate treated with thrombin, nearly all the VII1:Ag was inactive protein. At least 10% of patients with haemophilia possess inactive protein (Peake et al., 1979), and in the sample gel filtered it was not associated with vWf:Ag. All the antibodies mentioned in this work have been shown by Griffin (1986) to cross-react with VII1:Ag in the plasma of several mammalian species but not with these from horse, sheep or ox. The strongest reactions were with pig, baboon and rabbit plasmas. This strong cross-reaction with rabbit plasma led to this ELISA being used to measure rabbit VII1:Ag (Czerchawski et al., 1987). R Benson (personal communication, 1991) has indicated that a similar assay format may be used to determine VII1:Ag in canine plasma. Despite the difficulty in assigning absolute values to concentrates, the ELISA is entirely suitable for the assay of plasma VII1:Ag. It has a sensitivity of 0.006

u/ml with aninter-assaycoefficient ofvariationof6.3%. Forty-eight plasma samples were assayed by both ELISA and IRMA and the coefficient of correlation was 0.89. This ELISA is faster, more convenient and easier to perform than the IRMA which it has replaced in this laboratory. It is more accurate and the reagents, which are safer to handle, can be mass produced reliably. This laboratory used the assay during the calibration exercise for the second International Standard for VIII and vWf in plasma and the interassay coefficient of variation was reported as 2.84%. It can be concluded that this assay fulfils most of the practical targets that were set for this study and it is believed that it will be useful in both academic and clinical laboratories where VIII is studied. ACKNOWLEDGEMENTS The authors wish to thank the late D r Lisel Micklem and Professor Keith James for the production of monoclonal antibodies, Dr Nik Hunter for vWf:Ag assays and Miss Grace Middleton and Miss Yvonne Waterson for help in the initial studies. They would also like to thank Drs Joan Dawes, Brenda Griffin and Ron McIntosh for helpful advice. This work was supported, in part, by a grant K/MRS/50/C500 from the Scottish Home and Health Department to Drs K. James and D. B. L. McClelland. REFERENCES Bos, E.S., Van der Doelen, A.A., van Rooy, N. & Schuurs, A.H.W.N. (1981) 3,3’,5,5’-tetramethylbenzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme-immunoassay. Journal oflmmunoassay, 2, 187-204. Burke, R.L., Pachl, C., Quiroga, M., Rosenberg, S., Haigwood, N, Nordfang, 0. & Ezban, M. (1986) The functional domains of coagulation factor V1II:C. Journal of Biological Chemistry, 261, 12574-12578. Czerchawski, L., Hornsey, V.S.,Prowse, C.V. & Bessos, H. (1987) Cross-reactive factor VIII in the rabbit: Potential animal model for FVIII studies. Thrombosis Research, 48, 125-130. Dinesen, B. & Feddersen, C. (1983) An enzyme immunoassay (ELISA) for the quantitation of human factor VIII coagulant antigen (V1II:CAg). Thrombosis Research, 31, 707-718. Fass, D.N., Knutson, G.J. & Katzmann, J.A. (1982) Monoclonal antibodies to porcine factor VIII coagulant and their use in the isolation of active coagulant protein. Blood, 59, 594-600. Fulcher, C.A., Roberts, J.R., Holland, L.Z. & Zimmerman, T.S. (1985) Human factor VIII procoagulant protein. Monoclonal antibodiesdefine precursor-productrelation-

ELISA for VIII:Ag 229 ships and functional epitopes. Journal of Clinical Inuestigarion, 76, 1 17-1 24. Furlong, R.A., Chesham, J. & Peake, I.R. (1988) The combined use of monoclonal antibody-based enzymelinked immunosorbent assays (ELISA) for factor VIII antigen (V1II:Ag) and von Willebrand antigen (vWf:Ag) for the detection of carriers of haemophilia A. Clinicaland Loboratory Haematology, 10,295-305. Goodall, A.H. & Meyer, D. (1985) Registry of monoclonal antibodies to factor VIII and von Willebrand factor. International Committee on Thrombosis and Haemostasis. Thrombosis and Haemostasis, 54, 878-89 1. Griffin, B.D. (1986) Studies on Human Factor VIII. PhD Thesis, Council for National Academic Awards. Griffin, B.D., Micklem, L.R., McCann, M.C., James, K. & Pepper, D.S. (1986) The production and characterisation of a panel of ten murine monoclonal antibodies to human procoagulant factor VIII. Thrombosis and Haemostasis, 55,4046.

Hada, M., Yorifuji, H. & Arai, M. (1987) Analysis of the structure and function of factor VIII by monoclonal antibodies. Acta Haematologica Japonica, 50, 168 1-1 688. Hellstern. P., Miyashita, C., Kohler, M., Von Blohn, G., Kiehl, R., Biro, G., Schwerdt, H. & Wenzel, E. (1987) Measurement of factor VIII procoagulant antigen in normal subjects and in haemophilia A patients by an immunoradiometric assay and by an enzyme-linked immunosorbent assay. Hatmostasis, 17, 173-1 8 1. Holmberg. L., Borge, L., Ljung, R. & Nilsson, I.M. ( I 979) Measurement of anti-haemophilic factor A antigen (VIII: CAg) with a solid phase immunoradiometric method based on homologous non-haemophilic antibodies. Scandinavian Journal of Haematology, 23, 17-24. Hornsey, V.S. (1988) Studies on monoclonalantibodies to von Willebrandfactor and coagulationfactor VIfI. Ph D Thesis, Heriot-Watt University, Edinburgh. Hornsey, V.S.,Griffin, B.D., Pepper, D.S., Micklem, L.R. & Prowse, C.V. (1987) Immunoaffinity purification of factor VIII complex. Thrombosis and Haemostasis, 57, 102- 105. Hornsey, V.S.,, Waterston, Y.G. & Prowse, C.V. (1988) Artificial factor VIII-deficient plasma: Preparation using monoclonal antibodies and use in one-stage coagulation assays. Journal of Clinical Pathology, 41, 562-567. Ingerslev, J. & Stenbjerg, S. (1 986) Enzyme linked immunosorbent assay (ELISA) for the measurement of factor VIII coagulant antigen (CAg) using haemophilic antibodies. British Journal of Haematology, 62,325-332.

Lazarchick, J, & Hoyer, L.W. (1978) Immunoradiometric measurement of the factor VlII procoagulant antigen. Journal of Clinical Investigation, 62, 1048-1052. Muller, H.P.,vanTilburg,N.H., Derks, J., Klein-Breteler, E. & Bertina, R.M. (1981) A monoclonal antibody to VII1:C produced by a mouse hybridoma. Blood, 58, 1000-1006. Nakane, P.K. & Kawaoi, A. (1974) Peroxidase-labelled antibody. A new method of conjugation. Journal of Histochemistry and Cytochemistry, 22, 1084- 109 1. Nordfang, O., Ezban, M.& Dinesen, B. (1 985) Reactivity of factor VIII inhibitors in a micro ELISA for factor V1II:CAg and in solid phase immunoisolation of VIII: CAg. Thrombosis and Haemostasis, 53, 346-350. Peake, I.R., Bloom, A.L., Giddings, J.C. & Ludlam, C.A. (1979) An immunoradiometric assay for procoagulant factor VIII antigen: results in haemophilia, von Willebrand’s disease and fetal plasma and serum. British Journal o/ Haematology, 42, 269-28 I . Peake, I.R., Newcombe, R.G., Davies, B.L., Furlong, R.A., Ludlam. C.A. & Bloom, A.L. (1981) Carrier detection in haemophilia A by immunological measurement of factor VIIl related antigen (VIII RAg) and factor VIII clotting antigen (VIII CAg). British Journal of Haematology, 48, 65 1-660.

Reisner, H.M.. Barrow, E.S. & Graham, J.B. (1979) Radioimmunoassay for coagulant factor VIII-related antigen (VII1:CAg). Thrombosis Research, 14, 235-239. Rotblat. F., Goodall, A.H., O’Brien, D.P., Rawlings, E., Middleton, S.& Tuddenham, E.G.D. (1983) Monoclonal antibodies to human procoagulant factor VIII. Journal of Laboratory and Clinical Medicine, 101, 736-746. Rotblat, F. & Tuddenham, E.G.D. (1981) Immunologic studies of factor VIII coagulant activity (VI11:C). 1. Assays based on a haemophilic and an acquired antibody to VII1:C. Thrombosis Research, 21,431-445. Stel, H.V.,Veennan,E.C.I.,Huisman, J.G., Janssen.M.C.& van Mourik, J.A. (1983) A rapid one-step immunoradiometric assay for factor VIII-procoagulant antigen utilising monoclonal antibodies. Thrombosis and Haemostasis, 50, 860-863.

Tran. T.H. & Duckert, F. (1983) Dissociation of factor VIII procoagulant antigen VII1:CAg and factor VIII related antigen VIII R:Ag by EDTA-influence ofdivalent cation on the binding of V1II:CAg and V1II:RAg. Thrombosis and Haemostasis, 50, 547-55 1 .