The incorporation of polyethylene glycol (PEG) in pharmaceuticals has been a common strategy to improve drug properties. Use of ubiquitous PEG-containing drugs, foods, and cosmetics have borne anti-PEG antibodies in some individuals. Of particular interest are Anti-PEG IgE antibodies due to their potential role in hypersensitivity reactions. The accurate determination of these antibodies is critical for assessing the risk of allergic reactions. However, the measurement is complicated because IgE concentration in serum is low and necessitates a highly sensitive assay. In addition, assay specificity is key due to the high prevalence of other anti-PEG antibody isotypes which can interfere and obfuscate results and interpretations. To overcome these challenges, we have developed an electrochemiluminescent (ECL) assay that detects anti-PEG IgE in human sera with high sensitivity and enhanced specificity. Unique to our design strategy was to first select and isolate IgE antibodies via a bead-based enrichment procedure. Following enrichment, the samples are transferred to an MSD assay plate coated with PEGylated protein, which captures anti-PEG IgE antibodies. Ruthenylated anti-IgE antibody served to detect anti-PEG IgEs enabling the ECL signal. This anti-PEG IgE assay development demonstrates high sensitivity in sera (50 ng/mL). We measured specificity (interference tolerance) by evaluating the anti-PEG IgE detection in the presence of competing antibodies, which we term competing antibody threshold (CAT). The assay yielded a CAT of >250 molar excess of anti-PEG immunoglobulins such that the signal of anti-PEG IgE was still above the titer cut point. This design of experiment and level of sensitivity and specificity represents a significant bioanalytical advancement, providing a reliable new tool for monitoring anti-PEG IgE levels in patients. This talk will present our results, the bioanalytical approaches, the design strategy, and the data-driven decisions we made, the comparison to non-enrichment evaluations, as well as the challenges and opportunities in implementation of these technologies.
Learning Objectives:
Upon completion, participants will be able to understand the role anti-PEG Isotypes play in immunogenicity and the role anti-PEG IgE plays in hypersensitivity reactions.
Upon completion, participants will be able to recount benefit and cost considerations of antibody enrichment in the presented methodology.
Upon completion, participants will be able to critically evaluate the value and the potential of antibody isotype enrichment and its incorporation in other clinical assays.
Upon completion, participants will understand Competing Antibody Threshold (CAT) and role specificity impacts assessments of low abundant analytes like anti-PEG IgE.
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