SummaryNine human antibodies to factor VIII were isolated from haemophilic plasmas by affinity chromatography and gel filtration and six were subsequently subjected to immunological characterization. Three partially purified preparations were similarly characterized. Eight of the antibodies were characterized as being exclusively IgG and one preparation was found to contain IgM. Seven of the antibodies contained only a single light chain type, four being of type lambda and three of type kappa. Two antibody preparations contained both kappa and lambda light chains. In four of the preparations, only a single heavy chain sub-class could be demonstrated, three of IgG3 and one of IgG4. Of the remainder, three were a mixture of IgG3 and IgG4 sub-classes and one contained both IgG2 and IgG4. IgG sub-classification could not be achieved with the IgM-containing preparation. These results demonstrate a restricted heterogeneity of light and heavy chains in human antibodies to factor VIII.
SummaryAn immuno-affinity chromatography method was used to isolate human factor VIII and its antibodies and the mechanism of the affinity system was investigated using iodine labelling.Rabbit antibodies to human factor VIII were insolubilised onto CNBr — activated Sepharose 2B which was used for the preparation of affinity columns. Both VIII:C and VIIIR:Ag were adsorbed onto such columns from factor VIII preparations. The subsequent application of immunoglobulin preparations containing human antibodies to factor VIII resulted in the adsorption of these antibodies onto the columns. Adsorbed material was eluted from the affinity columns with 0.2 M glycine - HCl, pH 2.3.When 125I-labelled factor VIII and 131I-labelled human antibodies to factor VIII were used in this affinity system, the eluted material could be separated into three fractions by gel filtration on Bio-Gel A 1.5 m. Fraction 1 occurred at the void volume position, fraction 3 at a position corresponding to the elution position of IgG and fraction 2 at an intermediate position. 131I-labelled material was present in all three peaks. 125I-labelled material was present mainly in peak 1, with a little in peak 2. The results support the view that VIIIR: Ag, which binds heterologous antibodies, is non-covalently linked to a smaller subunit, VIII.C, which binds homologous antibodies.
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