A multiplex approach to isotyping antigen-specific antibodies using biotinylated antigen/streptavidin-phycoerythrin.
Analytical methods characterizing the immunogenicity of antigens are useful for monitoring, characterizing and predicting antibody responses to therapeutic biologics or vaccines. Distinct Luminex microspheres coupled with protein G, anti-human immunoglobulin (Ig)A, anti-human IgM and anti-human IgE were developed for the simultaneous capture of total IgG, IgA, IgM and IgE (IgGAME) antibodies from human or non-human primate serum. The fraction of antigen-specific antibodies captured on the beads was detected using biotinylated antigen/streptavidin-phycoerythrin. The method was demonstrated by isotyping antibodies directed against an anti-CD11a antibody therapeutic (RAPTIVA/efalizumab) from the serum of a cynomolgus monkey hyper-immunized with RAPTIVA over a 15-month period. The quantitative range of the antibody measurements, using 5 mul of sample, was determined to be 15 ng/ml to 50 mug/ml in 10% serum. By the use of any biotinylated antigen as a detector, this multiplexed isotyping assay can be broadly applied to human and non-human primate IgGAME immunogenicity studies.
McCutcheon
Krista
K
Antibody Engineering Department, Genentech Inc., South San Francisco, CA, USA.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D061067:Antibodies, Monoclonal, Humanized; D000941:Antigens / Q000737:chemistry*; D019921:Biotinylation; D006801:Humans; D007070:Immunoglobulin A / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007132:Immunoglobulin Isotypes / Q000032:analysis*; D007075:Immunoglobulin M / Q000276:immunology; D008252:Macaca fascicularis / Q000276:immunology; D008863:Microspheres; D010799:Phycoerythrin / Q000737:chemistry*; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000941:Antigens; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M; D010799:Phycoerythrin; D007073:Immunoglobulin E; D019809:Streptavidin; C472181:efalizumab