An electrochemiluminescence (ECL)-based assay for the specific detection of anti-drug antibodies of the IgE isotype

• We develop an IgE ADA assay for the detection of anti-drug IgE ADA in human serum.
• A drug-specific IgE chimeric mAb was generated and used as an ADA control.
• The biotinylated-drug and ruthenylated-omalizumab were used as antibody pairs.
• Utilization of a chimeric IgE mAb and omalizumab illustrates a general approach.
• The approach can be used for other biotherapeutics for the detection of IgE ADA.

To address a possible linkage between the occurrence of the hypersensitivity reactions and the induction of IgE anti-drug-antibodies (ADA), a drug specific IgE ADA assay was developed using electrochemiluminescence (ECL) technology. In the assay a drug-specific IgE isotype chimeric antibody was generated and used as an ADA positive control. The biotinylated drug X (an antibody) and ruthenium-labeled omalizumab (an anti-human IgE antibody) were used as capture and detection reagents, respectively.The binding affinities of the chimeric IgE isotype positive control have been shown to be highly comparable to drug X and drug Y (drug X is the 2nd generation of drug Y), indicating that it could serve as a highly useful control to compare and contrast the relative ability of the two generations of drug to elicit IgE ADA responses. The assay cut point factor (CPF) was estimated to be 1.13. The cut point factor derived from normal human serum samples was statistically equivalent to the cut point factor determined from targeted population samples. The assay could detect less than 250 ng/mL of IgE antibodies in the presence of 300 μg/mL drug X. The assay sensitivity was <0.2 ng/mL. A minimal prozone was observed at 100 μg/mL IgE ADA, but the sample remained highly detectable. The inter-assay precision was within 12%. The assay was not susceptible to non-specific matrix effects. The performance specifications ensured that the assay was suitable for validation. The combination of the chimeric IgE positive control and the detection antibody (ruthenium-labeled omalizumab) used for the assay could potentially provide a general bioanalytical approach for other biopharmaceuticals for the detection of IgE ADA responses.

Design of the biotin-drug/Sulfo-tag labeled omalizumab IgE ADA assay (a) and the biotin-drug/Sulfo-tag labeled drug (b) assay. Samples containing chimeric IgE control mAb were pre-incubated in the solution phase with a master mixture containing both the biotin-drug and Sulfo-tag-omalizumab or Sulfo-tag-drug conjugates. The complex was captured on a standard streptavidin-coated MSD plate, and a 2X MSD “T” read buffer is added to the wells. The plates are then read by the MSD Sector Imager 6000 reader.Figure optionsDownload as PowerPoint slide