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Solid‐Phase Immunoradiometric Assay of Factor‐VIII Protein
Author(s) -
Counts Richard B.
Publication year - 1975
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.1975.tb00878.x
Subject(s) - immunoradiometric assay , chemistry , chromatography , antibody , haemophilia a , serial dilution , elution , incubation , microgram , radioimmunoassay , microbiology and biotechnology , biochemistry , haemophilia , in vitro , immunology , biology , medicine , genetics , alternative medicine , pathology
S ummary . A solid phase immunoradiometric assay for factor‐VIII protein has been developed using 125 I‐labelled rabbit antibody made against highly purified factor VIII. Antibody specific for high molecular weight factor‐VIII protein was isolated using an immunoadsorbent consisting of highly purified factor VIII bound to diazotized m‐aminobenzyl (oxymethyl)‐cellulose. The purified anti‐factor VIII antibody was labelled with 125 I while bound to the immunoadsorbent and then eluted at pH 2.9. Dilutions of plasma samples for assay were incubated for 48 h in anti‐factor VIII antibody‐coated tubes. The tubes were washed to remove unbound proteins and the 125 I‐labelled, purified antibody was added. After 48 h incubation the tubes were washed to remove unbound antibody and counted. The concentration of immunoreactive factor‐VIII protein in pooled normal human plasma was determined to be 8 μg/ml. The minimum amount of factor VIII measured by the assay is less than 0.16 ng. Factor‐VIII protein was present in normal concentration in haemophilia A plasma, and in reduced concentration in von Willebrand's disease plasma. This method has the advantages of improved sensitivity, specificity, efficiency and economy as compared with previous factor‐VIII immunoassays.