A Radial Immunodiffusion Method for the Assay of Factor VIII:C Antigen (VIII:C Ag) in Plasma

Abstract

A method has been developed for the immunological quantitation of factor VIII:C Ag using a medium titre (50 new Oxford units) factor VIII:C antibody arising in a severe multitransfused haemophiliac. The method, which utilizes clotting inhibition in an agarose gel medium, gave close and significant correlation with the two-stage factor VIII:C procoagulant assay (r=0.83, P less than 0.01) for 54 normal subjects. Similar or higher values were found in 19 mild, moderate and severe haemophiliacs with 2-30% average normal plasma levels of factor VIII:C (r=0.72, P less than 0.01). Four mild von Willebrand patients gave similar results by immunoassay and procoagulant assay methods. A previously identified patient with cross reacting material (CRM+) gave an immunoassay within the normal range (66%) with only 4% VIII:C activity detectable. The method offers a simple, sensitive and apparently reliable procedure for the assay of plasma factor VIII:C Ag which may prove useful in the further investigation of factor VIII:C Ag and antibody heterogeneity. The procedure offers an alternative to immunoradiometric assay and may be of potential use in the assessment of the haemophilia carrier state and possibly the early detection of thrombosis.

The stability of VIII:C in frozen stored plasma samples-re-examined

“…Samples were first adsorbed with a one twentieth volume of 'Alhydrogel' aluminium hydroxide for 3 min at 3TC, and were then centrifuged at 2000 g for 5 min. VIII:C Ag was measured on 49 of the unadsorbed samples by the recently described agarose gel diffusion method (McLellan et al 1981). The latter method utilises an VIII:C antibody (currently 100 New Oxford Units), acquired in a severe haemophiliac, to measure the neutralizing capacity of serial plasma dilutions incorporated in an agarose gel medium.…”

“…It could be postulated that the stability of our samples reflects a greater stability of the concentrate VIII material present in haemophilic plasma, as compared to native plasma VIII. However, our observations on the storage of a pool of frozen normal plasma(McLellan et at. 1981) and recent observations on subsequent normal pools confirm little, if any loss of VIII :C activity during storage for up to 3 months at -35()C.…”

Influence of high molecular weight factor VIII on the measurement of low molecular weight factor VIII procoagulant in different assay systems

“…The assay will detect VIIIR: Ag down to 3% of normal plasma VIIIR: Ag. Only haemophilic plasmas determined to be negative for factor VIII procoagulant antigen (VII1C:Ag) (McLellan et al, 1981) were used in these studies.…”