A sensitive ELISA for von Willebrand factor (vWf:Ag)

Abstract

A sensitive and precise enzyme-linked immunosorbent assay for the measurement of the von Willebrand factor (new nomenclature:vWf:Ag, previous name: F VIII:RAg) of plasma and factor VIII concentrates was constructed using a microtitre plate based sandwich technique. The coating antibody extracted vWf:Ag from standard and test material. Bound fraction was detected by a F(ab')₂ peroxidase conjugate of the same antibody as used for coating. The sensitivity limit of the assay was 0.004 IU/ml, at least 10 times lower than the electro-immunoassay (EIA) using the same antibody, and better than any previously reported polyclonal ELISA for vWf.Ag. Imprecision between runs was 5.1% (CV%) at the 0.50 IU/ml level. Plasma from 30 healthy people gave 1.02±0.25 IU/ml (mean±1SD) in the ELISA assay and 0.99±0.30 IU/ml (mean±1SD) using electro-immunoassay (EIA) with a correlation coefficient of 0.967. Two cases of homozygous recessive von Willebrand's disease revealed vWf:Ag values at or below buffer blank in all dilutions. In 18 patients with type I von Willebrand's disease, a correlation coefficient of 0.899 was found comparing ELISA and EIA. All patients with variant von Willebrand's disease tested exhibited complete dose-response congruity of patient plasma and pooled standard plasma calibrated against the 1st International Standard for Plasma F VIII. Factor VIII concentrates also behaved like normal plasma in the assay.

Key words:

• ELISA
• von Willebrand's disease
• vWf:Ag