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 μl of sample, was determined to be 15 ng/ml to 50 μg/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.