Heterogeneity of autoantibodies to factor VIII: differences in specificity for apparently distinct antigenic determinants of factor VIII coagulant protein

The interference of antibodies to factor VIII coagulant protein (VIII:C) of 9 nonhemophilic patients with the binding to factor VIII coagulant antigen (VIII:CAg) of a reference hemophilic 125I-Fab' reagent, used in a liquid phase VIII:CAg assay, was studied. The binding competition was estimated from immunoradiometric assay (IRMA) dose-response slope of VIII:CAg present in patient plasma, interference of antibodies with the 125I-Fab' binding to VIII:CAg in normal plasma, and the displacement of antibody from the complexes with VIII:CAg by the 125I Fab'. Antibody populations from three patients were studied in detail; in the VIII:CAg assay, two of them interfered with the 125I- Fab' binding, and one did not (patient 1). The formation of stable complexes between antibodies of each patient and VIII:CAg was demonstrated by protein-A-Sepharose adsorption. The 125I-Fab' binding to VIII:CAg-anti-VIII:CAg IgG complexes indicated that patient 1 antibodies and the 125I-Fab' recognized different antigenic determinants, whereas the other two patient antibodies and 125I-Fab' recognized closely related or identical VIII:CAg determinants. These results demonstrate an apparently selective recognition of at least two distinct VIII:CAg determinants by naturally occurring antibodies, suggesting a possibility of a wider use of these antibodies in studies of the structure and function of factor VIII.     

Enzyme linked immunosorbent assay (ELISA) for the measurement of factor VIII coagulant antigen (CAg) using haemophilic antibodies

A method for the quantitation of factor VIII clotting antigen (VIII:CAg) has been developed based on a micro enzyme linked immunosorbent assay (ELISA) principle employing antibodies from two polytransfused haemophilia A patients. Solid polystyrene support bound IgG fraction of inhibitor plasma extracted VIII:CAg from normal plasma and samples. Bound VIII:CAg was detected by peroxidase labelled F(ab')2 fragment of the IgG used for solid phase. Two assays, each based on its particular inhibitor antibody, were set up. The F VIII clotting antigen in plasma of 30 healthy persons was found identical with the two VIII:CAg assays (r=0.97) and closely correlating with clotting activity (VIII:C) (r=0.84). Serum VIII:CAg was 67% (+/-14.5%) of the corresponding plasma value. In severe haemophilia A, 17 out of 19 had VIII:CAg values less than 1 U/dl. Two patients with cross-reactive material (CRM+) were found. In some milder cases of haemophilia A, higher values of VIII:CAg than VIII:C was recorded. The sensitivity of the method was 0.08 U/dl. Inter assay coefficient of variation at the 100 U/dl level was 9.5% (CV%), at the 2 U/dl level 16.4% (CV%). Mainly due to the great stability of enzyme conjugated antibody compared to the natural decay of radioiodinated material and subsequent loss of detecting material, ELISA was found superior to immunoradiometric assay (IRMA).     

Human factor VIII: a calcium-linked protein complex

The possible role of Ca2+ as an essential constituent part of the human factor VIII complex has been investigated by stability studies, metal determinations, and gel filtration experiments. In citrated plasma, the factor VIII coagulant activity (VIII:C) deteriorated during storage in a biphasic manner. Collection of blood in heparin, instead of chelating anticoagulants, or neutralization of citrate by addition of CaCl2 to heparinized citrate phosphate dextrose (CPD) plasma rendered VIII:C noticeably stable. At physiologic levels of ionized calcium, VIII:C was almost completely stable during incubation of plasma for 6 hr at 37 degrees C. The influence of other divalent ions was also studied. Highly purified factor VIII complex was subjected to atomic absorption spectrophotometric analysis and found to contain about 1.0 mole calcium per 220,000 daltons. This intrinsic calcium could be readily removed by EDTA. When heparin plasma and CPD plasma were chromatographed on Sepharose CL-6B at 37 degrees C, all the factor-VIII-related activities eluted together as large protein complexes. In contrast, factor VIII coagulant antigen (VIII:CAg) and factor-VIII-related antigen (VIIIR:Ag) were completely dissociated upon exposure to EDTA. From these observations it is concluded that human factor VIII circulates in normal plasma as a calcium-linked protein complex.     

Simplified immunoradiometric assay for factor VIII coagulant antigen

A simplified, non-competitive, solid phase immunoradiometric assay has been developed for the quantitation of factor VIII coagulant antigen (VIII:CAg)--the antigenic counterpart of FVIII coagulant activity (VIII:C). Both homologous and heterologous antibodies to human factor VIII (FVIII) were used in this assay. Initially, FVIII in a test sample was attached to immobilized, human IgG obtained from a polytransfused haemophilia A patient with a high titre antibody to VIII:C. The bound FVIII was then detected using rabbit 125I-IgG specific for human FVIII. The concentration of VIII:CAg correlated well with VIII:C levels in the plasma from normal donors (r = 0.84, n - 15). Homozygote von Willebrand's disease patients had undetectable levels of VIII:CAg in their plasma. Patients with severe haemophilia A (VIII:C less than 0.01 u/ml) could be divided into groups on the basis of the VIII:CAg levels, i.e. those having undetectable VIII:CAg and other with measurable VIII:CAg. VIII:CAg detected in normal serum was less than 0.002 u/ml. In this assay the use of human antibody to FVIII is considerably decreased compared to other methods for VIII:CAg, and the time-consuming steps to immunopurify human anti-FVIII antibody are eliminated.     

Assay of factor VIII antigen (VIII:CAg) in 294 Haemophilia A patients by a new commercial ELISA using monoclonal antibodies

Monoclonal antibodies (MoAbs 833 and D4H1) directed against human factor VIII (FVIII) have been produced on a large scale to measure VIII:CAg by two-site ELISA (Asserachrom VIII:CAg, Diagnostica Stago). F(ab’)₂ from MoAb 833 were used for coating and bound VIII:CAg was revealed with MoAb D4H1 coupled to peroxidase. Control plasma (100 VIII:CAg U dL^?1 by comparing with the International Standard) was used as reference. The assay sensitivity was 0.1 U dL^?1 VIII:CAg. No apparent effect of the plasma proteins was observed provided plasma dilution was 5. Thus this ELISA allowed us to estimate VIII:CAg levels of 0.5 U dL^?1 in plasma. Levels of VIII:CAg were similar to those of VIII:C (correlation coefficient r= 0.87) in plasma from normal individuals (32 cases) and in patients with von Willebrand disease of various types (30 cases). Among 294 patients with Haemophilia A (HA), 161 had severe HA (VIII:C < 1 U dL^?1). Among those patients, 124 were cross-reacting material (CRM) negative with undetectable VIII:CAg and 37 were CRM+ (VIII:CAg 1– 31 U dL^?1). In 42 patients with moderate HA (VIII:C 1– 5 U dL^?1), 33 were CRM reduced (VIII:CAg 0.5–8 U dL^?1) and nine were CRM+ with a VIII:CAg/VIII:C ratio of 6–91 (mean 34.3). In mild HA (91 cases with VIII:C 6 U dL^?1), 29 patients were classified as CRM+ (VIII:C 6–57 U dL^?1, VIII:CAg 17–130 U dL^?1 and VIII:CAg/VIII:C ratio 1.8–13.7 (mean 4.51)). In 62 CRM reduced patients there was a linear correlation between VIII:C (6–39 U dL^?1) and VIII:CAg (2–36 U dL^?1) levels (r= 0.88). In conclusion, this sensitive assay allows us to distinguish the quantitative CRM reduced and negative from the qualitative (CRM+) abnormalities in Haemophilia A.     

Normal pregnancy in a patient with a prior postpartum factor VIII inhibitor: with observations on pathogenesis and prognosis

A 22-yr-old primigravada developed a hemorrhagic diathesis 6 days after delivering a normal female infant and was found to have an immunoglobulin inhibitor of factor VIII (15 Bethesda units). The patient was treated with prednisone and the bleeding stopped soon thereafter. Her inhibitor titer decreased over the next 8 mo, at which time no inhibitor was detectable. Nine months later she became pregnant again and proceeded to have an uneventful pregnancy and delivery of a normal female infant without evidence of a recurrence of the inhibitor. Studies of 3H-thymidine incorporation into the patient's lymphocytes in the presence of her own plasma or plasmas from her husband, normals, a von Willebrand patient, and a hemophilic patient yielded equivocal results. Analysis of VIII:CAg using 2 different antisera failed to discern immunologic differences between the VIII:CAg of the patient, her husband, or her first child. A review of the literature revealed that there were no recurrences with second pregnancies in any of the 8 patients with postpartum factor VIII inhibitors whose inhibitors had completely disappeared prior to delivery. While the pathogenesis of this disorder remains uncertain, the apparently favorable prognosis for such patients should be considered in counselling with regard to future pregnancies.     

Factor VIII:C and VIII:CAg Response in Patients with Haemophilia A and von Willebrand's Disease after Administration of Different Factor VIII Concentrates or Plasma

S ummary . Factor VIII procoagulant activity (VIII:C) and factor VIII procoagulant antigen (VIII:CAg) were studied in seven patients with haemophilia A after administration of three different factor VIII concentrates or plasma. The in vivo recovery of VIII:CAg was less than that of VIII:C and the disappearance rate of VIII:CAg was much higher either when concentrates or plasma were given. The half-life of VIII:C was thus about 12 h but of VIII:CAg only about 3 h or less. Six patients with von Willebrand's disease were studied after administration of AHF- Kabi. In contrast to haemophilia A the discrepancy between VIII:C and VIII:CAg disappearance rates was not present in von Willebrand's disease, since both VIII:C and VIII:CAg showed a typical progressive increase. We conclude that factor VIII:C given to haemophilia patients does not behave like native VIII:C, not even when fresh plasma is used. Patients with von Willebrand's disease are capable of forming a normal VIII:C when appropriately stimulated.     

Precipitating anti-VIII:C antibodies in two patients with haemophilia A

S ummary . IgG from the plasmas of two haemophilia A patients with anti-VIII:CAg antibodies (1000 and 200 u/ml) was isolated and labelled with ¹²⁵I. The specific labelled anti-VIII:CAg IgG was further purified by binding to and elution from immobilized factor VIII/von Willebrand factor (F.VIII/vWF). When studied by immunodiffusion and autoradiography, both antibodies gave a precipitin line with normal plasma, serum, cryoprecipitate, purified F.VIII/vWF and the plasmas of two patients with haemophilia A⁺. No precipitin line was observed with the plasmas of 11 patients with haemophilia A⁻ or four patients with severe von Willebrand's disease. Levels of VIII:CAg obtained by radioelectroimmunoassay were in agreement with those obtained by immunoradiometric assay. This study demonstrates that, contrary to previous evidence, human anti-VIII:CAg antibodies are precipitating as well as neutralizing when studied by highly sensitive techniques.     

Degradation of factor VIII coagulant antigen by proteolytic enzymes

S ummary . The factors responsible for the lability of factor VIII coagulant activity (VIII:C) and factor VIII coagulant antigen (VIII:CAg) are poorly understood. In this study the VIII:C and VIII:CAg are studied after incubation with plasmin, trypsin or α-chymotrypsin. Both isolated human VIII:CAg and VIII:CAg associated with factor VIII-related antigen (VIII R:Ag) are evaluated. The antigenic sites of the VIII:CAg are somewhat more stable to the action of these enzymes than the functional activity, although both follow a generally parallel degradation. A biphasic decay curve is seen in the initial time points. No stabilization of the functional or antigenic reactivity is observed in the presence of the VIII R:Ag.
Lower concentrations of each enzyme cause an initial rise in the factor VIII:C in the presence of VIII R:Ag, but not in the isolated VIII:CAg. Higher concentrations of α-chymotrypsin cause activation of VIII:C and a slight decrease in the VIII:CAg values in both preparations. These enzymes may play a modulating role in the coagulation cascade through the activation and degradation of VIII:C and VIII:CAg.     

Factor VIII inhibitors: A clinical overview

There is much evidence to indicate that inhibitors to Factor VIII in patients with classical hemophilia are the result of an immunological response to exposure to material (VIII:C or VIII:CAg) that is absent or present in reduced amounts in these patients. The inhibitor is an antibody that is usually restricted in immunochemical composition and in many instances contains predominantly or exclusively γG3 or γG4 heavy chains. Exposure to Factor VIII in many inhibitor patients leads to typical anamnestic responses with marked increases in the level of the inhibitor. The tendency to develop inhibitors and the clinical characteristics of the inhibitor may be affected by genetic factors, basal levels of Factor VIII:C and/or VIII:CAg, and the nature and amount of the “immunizing” material. Currently accepted therapeutic modalities are aimed primarily at the management of acute bleeding episodes.     

Immunoblot cross-reactivity of factor VIII inhibitors with porcine factor VIII

Porcine factor VIII has been used successfully to treat factor VIII inhibitor patients whose plasmas have minimal cross-reactivity to porcine factor VIII. However, some inhibitor plasmas do inhibit porcine factor VIII, and the extent of procoagulant inhibition often increases after treatment with porcine factor VIII. Because there is no information about the porcine factor VIII epitopes with which these antibodies react, we have compared the immunoblot and enzyme-linked immunosorbent assay (ELISA) reactivities with porcine and human factor VIII for 20 inhibitor plasmas (11 from hemophilia A patients and 9 autoantibodies). Immunoblots identified binding to porcine factor VIII for only 2 of the 12 plasmas from patients who had not received porcine factor VIII, but this reactivity could not be predicted from the inhibitor titer to porcine factor VIII. Immunoblot reactivity with porcine factor VIII was detected for 7 of 8 inhibitor plasmas from patients who had been previously treated with porcine factor VIII, and the strength of this reactivity was generally related to the inhibitor titer. Of the 5 plasmas that were immunoblot positive with the porcine factor VIII A2 domain, 4 had inhibitor titers greater than 45 Bethesda units when tested with porcine factor VIII, whereas only 1 of 15 of the other plasmas had this level of inhibitor activity with porcine factor VIII. In contrast, immunoblot reactivity to the porcine factor VIII A1 domain did not correlate with the antiporcine VIII inhibitor titer. We also determined the effect of preincubation with human or porcine factor VIII on immunoblot reactivity. In one case, immunoblot reactivity with porcine factor VIII was absorbed with porcine, but not human, factor VIII, which is consistent with antibody formation after treatment with porcine factor VIII. In no cases did human factor VIII reduce the reactivity of inhibitor plasmas with the porcine A1 domain, suggesting that these antibodies are directed at unique porcine factor VIII determinants. The reactivity to porcine A2 in 2 plasmas probably represented cross-reactivity of similar A2 determinants, because it was absorbed by both human and porcine factor VIII. Although the ELISA assays with porcine factor VIII detected antibodies in some plasmas that could not be identified by inhibitor assay or immunoblot, the level of ELISA reactivity was generally consistent with the titers of the other assays.     

Synthesis and Release of Factor VIII by Cultured Human Endothelial Cells

Endothelial cells (ECs) derived from human umbilical veins were cultured in order to study the physiological control of factor VIII synthesis and release. The culture media were studied from multiple replicate cultures at confluence. Factor VIII related antigen (VIIIR:Ag) and factor VIII coagulant antigen (VIII:CAg) were measured by sensitive immunoradiometric assays. De novo synthesis of factor VIII related protein (VIII:R) was quantitated by incorporation of labelled amino acids into specific protein subunits. The following agents were added to the culture medium in a range of concentrations from physiological to pharmacological: adrenaline, 5 hydroxytryptamine, 2,3-DPG, cyclic AMP, thyroxine, hydrocortisone, and human growth hormone. None of them had any effect at any concentration on the rate of accumulation of VIIIR:Ag in the culture medium. Addition of exogenous factor VIII had no effect on do novo synthesis of VIII:R. VIII:CAg was found to be stable under the conditions of culture but none was released from the ECs. Long-term monocyte cultures also failed to release VIII:CAg. It appears that VIII:R is a constitutive gene product of umbilical vein endothelial cells and that VIII:CAg is not made by these cells.     

The Release of Plasminogen Activator and Factor VIII after Injection of DDAVP in Healthy Volunteers and in Patients with von Willebrand's Disease

Increases in plasma concentrations of VIII:C, VIII:CAg, VIIIR:Ag and plasminogen activator (PA) were observed in 50 healthy volunteers given i.v. injections of DDAVP (desaminocys¹-8-D-arg-vasopressin). The PA activity reached its maximum immediately after the injection, VIII:C, VIII:CAg and VIIIR:Ag after 3040 min. However, a positive correlation was found when the PA and VIII:C responses in each of the normals were analysed. DDAVP was also administered to 3 patients with severe von Willebrand's disease. 2 of the patients displayed no changes in VIII:C, VIII:CAg, VIIIR:Ag or VIIIR:RCF and there was no increase in PA. The third patient responded with an increase in VIII:C and to a minor degree in VIII:CAg. This patient developed fibrinolytic activity, but in the lower normal range. In 3 other patients with mild von Willebrand's disease DDAVP caused increases in VIII:C, VIII:CAg, VIIIR:Ag and PA. We feel that the combined data may support the concept that one and the same target cell is involved in the DDAVP mediated release of factor VIII related activities and PA.     

Apparent molecular weight of purified human factor VIII procoagulant protein compared with purified and plasma factor VIII procoagulant protein antigen

We have compared apparent molecular weights of purified factor VIII procoagulant protein (VIII:C) and VIII:C antigen (VIII:CAg) by two different NaDodSO4 gel electrophoretic techniques. In a discontinuous NaDodSO4-7.5% polyacrylamide system, reduced and unreduced VIII:C, purified from commercial factor VIII concentrates by a monoclonal antibody immunoadsorption technique, showed a major doublet at mol wt 0.79 and 0.8 X 10(5) and less intense bands extending up to 1.9 X 10(5). In NaDodSO4-4% polyacrylamide/0.5% agarose gels (NaDodSO4-4% PAAGE), purified VIII:C had a major band of mol wt 1.0 X 10(5), with minor bands of mol wt 0.96, 1.1, 1.4, 1.6, 1.8, 2.2, and 2.4 X 10(5). In NaDodSO4-4% PAAGE of 125I-anti-VIII:C-Fab-VIII:CAg complexes, the major and minor forms of VIII:CAg in purified VIII:C had the same molecular weight as above when calculated by subtracting the molecular weight of 125I-Fab from 125I-Fab-VIII:CAg. In both plasma and factor VIII concentrate, a band of mol wt 2.4 X 10(5) predominated, and minor VIII:CAg forms of mol wt 2.6, 1.8, 1.2 and 1.0 X 10(5) were also visible. We conclude that the molecular weight of plasma VIII:CAg forms agree with those derived from protein stains of purified VIII:C in the NaDodSO4-4% PAAGE system, but that consistently lower molecular weight values are obtained for purified VIII:C in the discontinuous system. Both native and either disaggregated or proteolyzed VIII:C species are present in the purified VIII:C preparation.     

Analysis of factor VIII coagulant antigen in normal, thrombin-treated, and hemophilic plasma.

The relationship between Factor VIII coagulant antigen (VIII:CAg) and Factor VIII-associated von Willebrand factor (VIII:vWF), and the effect of thrombin on VIII:CAg have been determined in plasma by using complexes of VIII:CAg and 125I-labeled human anti-VIII:CAg-Fab. Antibody-treated plasma samples were electrophoresed on NaDodSO4/polyacrylamide agarose gels and analyzed by autoradiography. The major VIII:CAg-125I-labeled Fab complex that persisted in NaDodSO4 had Mr 3.2 x 10(5). This Mr value was confirmed by column chromatography and sucrose density centrifugation and is presumed to reflect a free VIII:CAg of Mr 2.7 x 10(5). Minor bands were also present on autoradiograms of normal plasma corresponding to Mr values of 2.5, 1.85, and 1.7 x 10(5) (free VIII:CAg related proteins with Mr values of 2.0, 1.35, and 1.2 x 10(5), respectively). None of the VIII:CAg bands was present in plasma samples from five patients with severe hemophilia A. No radioactivity was associated with VIII:vWF multimers on NaDodSO4 gels. Thrombin treatment of normal plasma eliminated the radioactive band at 3.2 x 10(5) and increased the intensity of a band of Mr 1.7 x 10(5). Generation of this presumed VIII:CAg fragment of Mr is approximately equal to 1.2 x 10(5) coincided with a thrombin-induced increase in Factor VIII coagulant activity. These data demonstrate that the form of VIII:CAg detected in normal plasma is not covalently linked to VIII:vWF multimers and is absent in plasma from five hemophilia A patients. Thrombin-induced proteolysis of VIII:CAg can be detected in microliter quantities of normal plasma.     

The combined use of monoclonal antibody- based enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII: Ag) and von Willebrand factor antigen (vWF: Ag) for the detection of carriers of haemophilia A

Enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII: Ag) and von Willebrand factor antigen (vWF: Ag) have been developed, each employing monoclonal antibodies. In the majority of severe haernophilic plasmas tested, VIII: Ag was undetectable by ELISA and also by immunoradiometric assay (IRMA) using haemophilic VIII:C antibodies. In haemophilic plasmas with mild/moderate deficiency of coagulant factor VIII (VIII: C), there was no significant difference between the two immunoassays although there was a general trend for ELISA VIII: Ag results to be higher. Assay of von Willebrand's disease (vWd) plasmas with the ELISA for vWF: Ag demonstrated reduced levels of this antigen in type I vWd, normal levels in type IIA, and a severe reduction of vWF:Ag in type III vWd. The discrimination of obligate carriers of haemophilia from normal was determined using ratios of factor VIII/vWF. Factor VIII antigen/von Willebrand factor antigen measured by IRMA and Laurell immunoelectrophoresis respectively, gave a superior discriminant to that of VIII: C/vWF: Ag (Laurell), but optimal discrimination was obtained with the combination of ELISAs for VIII: Ag and vWF: Ag.     

The effect of liver disease on factors V, VIII and protein C

The components of the factor VIII complex were estimated by immuno- and bioassays in 85 patients with liver disease. The plasma concentrations of the antigens were elevated in 65% (VIII:CAg) and in 76% (VIIIR:Ag) of patients while the biological activities were elevated in only 14% (VIII:C) and 15% (VIII:RiCof). There was no correlation with C-reactive protein, used as a measure of an acute phase reaction (X2 = 0.7; P = 0.1); or with severity of liver disease as judged by prothrombin ratio (P = 1.0) but highest values were observed in patients with cholestatic liver disease. Following parenteral vitamin K there was a significant fall in both the biological activity of VIIIC (36%) and of VIII:CAg (38%) in 13 vitamin K deficient patients (P less than 0.001) but no change in 23 vitamin K replete patients or in the VIIIR:Ag levels in either group. Factor V levels were lower in patients with parenchymal liver disease (0.54 +/- 0.1 units/ml, mean +/- SEM, n = 12; normal range 0.5-1.5 units/ml) than in patients with extrahepatic cholestasis who were vitamin K deficient (1.2 +/- 0.1 units/ml, P less than 0.0001). The levels of protein C antigen, the vitamin K dependent protease which inactivates factors VIII:C and V, was at the lower end of the range in both groups (0.7 +/- 0.1, mean +/- SEM, n = 18, normal range 0.74-1.4 units/ml). There was no significant change in either protein C antigen or factor V following vitamin K. The discrepancy between the biological activity of factor VIII and the antigen levels could represent accumulation of partially degraded factor VIII or production of a hypoactive form. There is no evidence that the reduction in VIIIC and VIII:CAg following vitamin K was mediated by protein C.     

Isolation of Human Antibodies to Factor VIII

It has been claimed that human anti-VIII: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-VIII:C antibodies appearing in haemophiliac patients following replacment 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-VIII:C antibodies were recovered following filtration of the antigen-antibody complexes on Biogel A-5m, dissociation of complexes at pH 3.5 and final isolation by filtration on Sephadex G-200. In a solid phase system, the same IgG fraction was specifically bound to insolubilized human factor VIII. Purified anti-VIII:C antibodies were subsequently recovered by elution of antigen-antibody complexes with magnesium chloride. The results demonstrated that stable complexes from between anti-VIII:C antibodies and either the whole factor VIII molecule, or VIII: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.     

The combined use of monoclonal antibody-based enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII:Ag) and von Willebrand factor antigen (vWF:Ag) for the detection of carriers of haemophilia A

Enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII:Ag) and von Willebrand factor antigen (vWF:Ag) have been developed, each employing monoclonal antibodies. In the majority of severe haemophilic plasmas tested, VIII:Ag was undetectable by ELISA and also by immunoradiometric assay (IRMA) using haemophilic VIII:C antibodies. In haemophilic plasmas with mild/moderate deficiency of coagulant factor VIII (VIII:C), there was no significant difference between the two immunoassays although there was a general trend for ELISA VIII:Ag results to be higher. Assay of von Willebrand's disease (vWd) plasmas with the ELISA for vWF:Ag demonstrated reduced levels of this antigen in type I vWd, normal levels in type IIA, and a severe reduction of vWF:Ag in type III vWd. The discrimination of obligate carriers of haemophilia from normal was determined using ratios of factor VIII/vWF. Factor VIII antigen/von Willebrand factor antigen measured by IRMA and Laurell immunoelectrophoresis respectively, gave a superior discriminant to that of VIII:C/vWF:Ag (Laurell), but optimal discrimination was obtained with the combination of ELISAs for VIII:Ag and vWF:Ag.     

Immunoradiometric Assay of Factor VIII: Coagulant Antigen using Four Human Antibodies. Study of 27 Cases of Haemophilia A

Immunoradiometric assay (IRMA) of VIII:C antigen was performed using either IgG or monovalent Fab fragments from four antibodies arisen in polytransfused haemophilia A patients (titre between 100 and 1500 U/ml). Using IgG isolated by a solid or a liquid phase system, only the high titre (greater than or equal to 1000 U/ml) antibodies could be used for IRMA, with a sensitivity of 0.2% VIII:CAg. Using Fab fragments isolated by liquid phase, high and low (less than or equal to 150 U/ml) titre antibodies could be used and the IRMA was significantly improved with a 10-fold higher sensitivity. The affinity of the antibodies for VIII:CAg, studied by displacement curves using a modification of the IRMA, was found not to depend upon the titre of the antibody. Comparative levels of VIII:C and VIII:CAg in 27 cases of haemophilia A emphasize the heterogeneity of this disorder, two types of severe and three types of mild haemophilia being observed.