Vendor-specific HRP antibody conjugate differences lead to unexpected immunoassay reagent interactions.

Aim: An interaction observed in an anti-drug antibody (ADA) bridging ELISA between the drug and the anti-digoxin (aDIG) conjugated horseradish peroxidase (HRP) led to increased assay background across several commercial aDIG-HRP sources. Understanding the source of this interaction will help inform reagent qualities to be considered in production, evaluation, and selection for bioanalytical assays.

Methods and materials: This work evaluates commercial sources of aDIG-HRP through bridging ELISA, binding ELISA, SPR, and chromatography to determine what characteristics of aDIG-HRP lead to the interaction observed in a bridging ELISA.

Results: Three of four commercial aDIG-HRP sources evaluated exhibit drug binding and size heterogeneity with large (>250 kDa) species. SE-UHPLC of one source localizes this binding capability to the >6 MDa species. Under typical HRP conjugation conditions, HRP can conjugate to itself, and these larger species can bind to drug.

Conclusion: The conjugation of HRP to aDIG may lead to large species linked to HRP self-conjugation that can interfere with bioassays if not carefully controlled. Therefore, it is important to evaluate commercial reagents for consistency, availability, and critical quality when incorporating a new lot/vendor for better understanding of unexpected assay performance and effectively support reagent and assay life cycle management.