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).     

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.     

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.     

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.     

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.     

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.     

Coagulation Factors in the Human Fetus of about 20 Weeks of Gestational Age

S ummary In plasma samples of 11 fetuses of about 20 weeks of gestational age the following coagulation factors have been determined (mean values found are given in parentheses): fibrinogen (0·30 U/ml), prothrombin (±0·17 U/ml), factor V (0·28 U/ml), factor VII (0·21 U/ml), factor VIII coagulant activity (factor VIII:C) (0·12 U/ml), factor VIII-related antigen (factor VIIIR:Ag) (1·04 U/ml), coagulant factor VIII-related antigen (factor VIII:CAg) (0·19 U/ml), factor IX coagulant activity (0·05 U/ml), factor IX antigen (≤0·03 U/ml), factor X (0·19 U/ml) and antithrombin III (AT-III) (0·23 U/ml).
Our data support the evidence that prenatal diagnosis of haemophilia A is at present possible; less optimism is warranted where haemophilia B is concerned. The number of samples is sufficient to establish normal values for the age group.
Means of quality control of the sample—which is often difficult to obtain—are discussed.     

An ELISA assay for the detection of factor VIII antibodies - comparison with the conventional Bethesda assay in a large cohort of haemophilia samples

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure factor VIII antibodies in haemophilia patients. The assay utilizes binding of the antibodies in the plasma to solid phase antigen, i.e. recombinant factor VIII which was subsequently detected by a human polyclonal IgG labelled with the alkaline phosphatase-p-nitrophenyl phosphate substrate system. Comparisons were made with the Bethesda assay for the quantitation of factor VIIII inhibitors. Dose response curves for the reference standards were consistently linear and reproducible. The assay was specific for factor VIII antibodies, showing a negative reaction for antibodies to other coagulation factors, antinuclear factors and antiphospholipid antibodies. Using this method, 312 samples from haemophilia A patients and 31 samples from healthy controls were screened for the presence of inhibitors and compared with the conventional Bethesda assay. Twenty-four cases were found to be positive for inhibitors in both the ELISA and Bethesda assay. Five additional cases were also found to be positive in the ELISA assay, which, however, were negative in Bethesda assay. One patient who was initially positive for factor VIII inhibitors both by the Bethesda assay and ELISA eventually became negative for the Bethesda assay (<0.5 BU/ml) but was still positive for the ELISA assay. The ELISA thus described had a specificity of 97.8% and a sensitivity of 100% when tested against a large cohort of haemophilia A samples.     

Factor VIII Clotting Antigen (VIIICAg) in Haemophilia Measured by Two Immunoradiometric Assays (IRMA) using Different Antibodies, and the Measurement of Inhibitors to Procoagulant Factor VIII (VIIIC) by IRMA

Factor VIII clotting antigen (VIIICAg) was measured by immunoradiometric assay (IRMA) using two different antibodies. Both antibodies arose in polytransfused severe haemophiliacs and had similar titres against VIIIC. In 12 normal plasmas there was no significant difference in VIIICAg values obtained (VIIICAg (AbI) = VIIICAg (AbII)). In the majority of 15 severe haemophiliacs tested VIIICAg was undetectable by both antibodies. In 28 mild to moderate haemophiliacs VIIICAg (AbII) was significantly greater than VIIICAg (AbI) ( P < 0·01) suggesting different antigenic determinants. The difference, however, was small and does not affect diagnosis of haemophilia. A modified IRMA has been used to measure and VIIIC inhibitors by competition of the inhibitor with ¹²⁵I labelled VIIICAg antibodies for common antigenic determinants. Using an inhibitor of 225 Bethesda units as a standard, results by IRMA of inhibitors in severe haemophiliacs have been similar to those obtained by clotting assay, but with a sensitivity of 0·01 u/ml suggesting the possible use in the detection of weak inhibitors.     

Heterogeneity of haemophilia A: a study with three different antisera

One human and one rabbit antibody against VIII:C--in a fluid-phase, inhibitor neutralization assay (INA)--and one human antibody--in a solid-phase, immunoradiometric assay (IRMA)--were used to investigate a group of 59 patients with severe, moderate and mild haemophilia A. Patients were classified as haemophilia A+ when the VIII:C/VIIIAG ratio was less than 0.4 while the absolute VIII:C antigen (VIIICAG) value exceeded 0.25 u/ml. Three haemophiliacs (from two pedigrees) were classified as A+ in all three assay systems. 50% of the patients were classified as haemophilia A+ on the basis of at least one assay. The other 50%, including 66% of the patients with severe haemophilia, were shown to be A- by all three assay systems. Combining the data obtained with the three different antibodies four different categories could be distinguished, in addition to the classification based on severity.     

Response to infusions of polyelectrolyte fractionated human factor VIII concentrate in human haemophilia A and von Willebrand's disease

S ummary . Factor VIII was purified from cryoprecipitate by ion exchange chromatography on solid phase polyelectrolyte E-5 (PE-E5). The product was highly purified (3.5 u VIII: C/mg protein) compared to conventional concentrate (0.3 u VIII: C/mg protein) with low fibrinogen, low isoagglutinin titre, and a ratio of factor VIII coagulant activity (VIII: C) to factor VIII related antigen (VIIIR: Ag) of 16:1.
Trial infusions of this material (PE VIII) were given to three patients with severe haemophilia A and one patient with homozygous von Willebrand's disease. These patients also each received separate infusions of intermediate purity concentrate (IPC) for comparison. There were no adverse effects. The mean half life of VIII: C after PE VIII infusion in the haemophiliacs was 10.9 h and after IPC was 12.1 h, a statistically insignificant difference. The survival of factor VIII coagulant antigen (VIII:CAg) was similar to that of VIII:C. In contrast, the half life of VIII:C and of VIII:CAg was very short after infusion of PE VIII in the patient with von Willebrand's disease (2.4 h). IPC when infused in this patient produced a typical secondary rise of VIII:C. Two bleeding episodes in severe haemophiliacs were satisfactorily treated with PE VIII. PE-E5 deserves further study as a means of preparing clinical concentrates of factor VIII.     

Isolation of Fab and Fc Fragments from a Plasmin-Treated Human Immunoglobulin by High-Speed Gel Filtration on TSK G3000SW and G3000SWG

A method is described for isolating intact 7S IgG, Fab and Fc fragments from a plasmin-treated immunoglobulin by high-speed gel filtration on a TSK G3000SW or G3000SWG column. The isolated 7S IgG, Fab and Fc fragments reacted with antihuman IgG, Fab and Fc antiserum, respectively, in both Ouchterlony's double immunodiffusion and immunoelectrophoresis. Each of the Fab and Fc fragments formed a single precipitation line, demonstrating their homogeneity. Anticomplementary activities of intact 7S IgG, Fab and Fc fragments were 2.5, less than or equal to 10 and 2.5 mg protein/ml to inhibit 2 units of CH50, respectively, and the diphtheria antitoxin contents were 2.1, 6.0 and 1.5 IU/150 mg protein, respectively. The molecular composition of plasmin-treated immunoglobulin as determined by gel filtration on a TSK G3000SW column was as follows: 7S IgG 38.0 +/- 0.2%; Fab 42.6 +/- 0.1% and Fc fragment 19.3 +/- 0.1%.     

Detection of factor IX inhibitors by immunoradiometric assay

An immunoradiometric assay (IRMA) for the determination of factor IX inhibitors in haemophilia B was developed. The assay was based on competitive binding between radiolabelled anti-IX and inhibitors in the test material of immobilized IX:C. 5 haemophiliacs with known inhibitors were investigated with the new method and with a conventional neutralization test. An inhibitor titre as low as 5 X 10(-4) U/ml could be measured by IRMA, showing it to be about 500 times as sensitive as the conventional neutralization assay used, and in 2 cases the inhibitors could only be detected by IRMA. The new method is thus useful for the investigation of patients with low inhibitor titres, or when inhibitors with divergent properties are suspected.     

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.     

Absence of anamnestic response after transfusion of washed red blood cells in haemophilia A patients with antibody to factor VIII

Washed red blood cells (WRC) have been used for replacing the blood loss in haemophilia A patients with antibody to Factor VIII. Levels of VIII coagulant activity (VIII:C), VIII coagulant antigen (VIII:CAg) and VIII related antigen (VIIIR:Ag) have been measured during the different steps of the preparation of WRC. The amount of VIII:CAg decreases very rapidly after washing and both are undetectable in the final product. These results were correlated with the absence of anamnestic response in 10 haemophilia A patients with inhibitor known as high responders.     

Analysis of factor VIII inhibitors in a haemophilia A patient with an Arg593-->Cys mutation using phage display

We characterized anti-factor VIII antibodies in a mild haemophilia A patient with an Arg593-->Cys mutation in the A2 domain, using V gene phage-display technology. All isolated single-chain variable-domain antibody fragments were directed against residues Arg484-Ile508, a binding site for factor VIII inhibitors in the A2 domain. After a further period of replacement therapy, a transient rise in inhibitor titre was observed. These antibodies were directed against the A2 domain. Activation of a pre-existing pool of B cells, which express antibodies against residues Arg484-Ile508, could explain the rapid anamnestic response.     

Studies on the stability of factor VIII coagulant antigen (VIII: CAg) in the presence of VIII: C antibodies

S ummary . The stability of factor VIII coagulant antigens (VIII:CAg) at 56°C was investigated using an immunoradiometric assay for VIII: CAg. In normal or CRM⁺ haemophilic plasmas VIII: CAg was rapidly inactivated at 56°C. VIII: CAg in spontaneous VIII: C inhibitor plasmas and in post-treatment samples from haemophiliacs with VIII: C inhibitor was resistant to inactivation at 56°C, indicating the presence of heat stable VIII: CAg-anti VIII: CAg complexes.
In vitro VIII: CAg-anti VIII: CAg complexes were formed by incubation of diluted VIII: C antibodies and normal plasma and the stability of these complexes at 56°C was studied. Haemophilic VIII: CAg antibodies formed heat stable immune complexes over a narrow range of inhibitor dilutions whilst some spontaneous VIII: CAg antibodies formed these stable complexes over a much wider range of dilutions emphasizing the difference in the properties of these antibodies.     

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.     

Immunoradiometric Assay of Factor VIII Related Antigen, with Observations in 32 Patients with von Willebrand''s Disease

A solid phase non-competitive immunoradiometric assay (IRMA) has been developed which allows measurement of factor VIII related antigen (VIIIR:AG) levels in normal plasma as low as 2.5 x 10(-4) U/ml. The assay is based on the extraction of VIIIR:AG from test plasma by means of polystrene tubes coated with a specific unlabelled anti-VIIIR:AG rabbit antiserum and subsequent labelling of the extracted antigen with 125I-labelled anti-VIIIR:AG rabbit IgG.     

A longitudinal evaluation of anti‐FVIII antibodies demonstrated IgG4 subclass is mainly correlated with high‐titre inhibitor in haemophilia A patients

The development of inhibitory antibodies against factor VIII (FVIII) (inhibitor) is the major complication in haemophilia A patients. The FVIII‐binding antibodies development comprises a polyclonal immunoglobulin (Ig) G response. Recent studies showed strong correlation between the presence of neutralizing anti‐FVIII antibodies (inhibitors) and IgG4 subclass. The aim of this study was to evaluate anti‐FVIII IgG subclasses in haemophilia A patients with inhibitor both in a cross‐sectional and in a longitudinal analysis. Inhibitors were determined by Nijmegen–Bethesda assay. Anti‐FVIII IgG subclasses were performed by ELISA, and samples from 20 healthy individuals were used to validate the test. We studied 25 haemophilia A patients with inhibitor, previously treated exclusively with plasma‐derived FVIII concentrates or bypassing agents. The IgG subclasses distributions were evaluated in two groups of patients classified according to inhibitor response. IgG1 and IgG4 antibodies were most prominent in haemophilia A patients with inhibitors when compared with IgG2 and IgG3. This study reports for the first time the behaviour of FVIII‐binding IgG1 and IgG4 subclasses in a longitudinal analysis, in a clinical setting, of high‐response inhibitor haemophilia A patients, showing the correlation of IgG4 and the inhibitor titres. In spite of being considered a non‐pathologic antibody subclass with anti‐inflammatory properties in other situations, IgG4 is correlated with the presence of high‐titre inhibitor in the haemophilia setting. The comprehension of the IgG4 role in immune response may be crucial to establish the process for designing specific tolerance to FVIII.