Competitive enzyme-linked immunoassay of monoclonal immunoglobulin G anti-D preparations

The development of monoclonal immunoglobulin G (IgG) anti-D for prophylaxis necessitates estimation of their potency in terms of their red cell-binding ability, but it is unclear which quantification methodology is most suitable for this. The aim of this study was to assess 50 monoclonal anti-D from the 4th International Workshop for quantitative and qualitative binding to red cells in a competitive enzyme-linked immunoassay (EIA) in which varying amounts of monoclonal antibodies (MoAbs) and a constant amount of biotinylated monoclonal anti-D (BRAD-5) compete for red cell binding. The potencies of the MoAb were estimated against the International Reference Preparation (IRP) for anti-D Ig. Two MoAbs as supplied were insufficiently inhibitory of biotinylated BRAD-5 binding to be quantified; the potencies of the remainder ranged from 4 to 343 IU mL(-1) and included 11 MoAbs showing dose-responses that were nonparallel to those of other MoAb and the IRP. Estimation of the concentration of antibody in the supernatants by radial immunodiffusion ranged from 0.5 to 61 mug mL(-1) , giving specific activities of <1-24 IU mug(-1) . The results show that competitive EIA is suitable for quantitating most monoclonal anti-D for development and quality control purposes, regardless of their D epitope reactivity.