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.
Shetty
Shrimati
S
Institute of Immunohaematology (ICMR), KEM Hospital, Parel, Mumbai, India.
Ghosh
Kanjaksha
K
Mohanty
Dipika
D
D016022:Case-Control Studies; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D005169:Factor VIII / Q000276:immunology*; D006467:Hemophilia A / Q000276:immunology*; D006801:Humans; D007518:Isoantibodies / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D007518:Isoantibodies; D005169:Factor VIII