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Methods Mol Biol. 2008;418:187-208.

A multiplex approach to isotyping antigen-specific antibodies using biotinylated antigen/streptavidin-phycoerythrin.

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Antibody Engineering Department, Genentech Inc., South San Francisco, CA, USA.


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.

[Indexed for MEDLINE]

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