title: Competitive enzyme-linked immunoassay of monoclonal immunoglobulin G anti-D preparations.
abstract: 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 micro g mL-1, giving specific activities of <1-24 IU micro g-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.
authors.affiliation: National Institute for Biological Standards and Control, Potters Bar, Herts, UK. sthorpe@nibsc.ac.uk
authors.forename: S J
authors.initials: SJ
authors.lastname: Thorpe
authors.forename: B
authors.initials: B
authors.lastname: Fox
authors.forename: C
authors.initials: C
authors.lastname: Turner
authors.forename: M
authors.initials: M
authors.lastname: Scott
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis*; D000915:Antibody Affinity; D001667:Binding, Competitive; D019921:Biotinylation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes; D004899:Erythroblastosis, Fetal / Q000517:prevention & control; D004912:Erythrocytes / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D007518:Isoantibodies / Q000032:analysis*; D018029:Rho(D) Immune Globulin
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D007074:Immunoglobulin G; D007518:Isoantibodies; C061961:RHO(D) antibody; D018029:Rho(D) Immune Globulin