Optimized antibody labeling efficiency through continuous centrifugal diafiltration (TECH1P.838)

Abstract

Abstract Immuno-detection encompasses a range of analytical methodologies. These assays, which quantify and/or localize expression of protein(s) in complex samples, are reliant upon tagged target-specific antibodies. While a range of labeling kits is available, the workflow is hampered by multiple dialysis-based buffer exchange steps that are both time-consuming and subject to sample loss. Also, due to differences between labeling targets (and dyes), prior determination of labeling reaction parameters must occur. We present an alternative method for small-scale protein labeling based upon continuous flow diafiltration. By exploiting the rapid kinetics of labeling reactions, continuous diafiltration minimizes reaction time and exposure to excess dye, guaranteeing maximal target labeling and thus eliminating the need for up-front optimization of reaction conditions. All phases of the conjugation workflow can be performed in a single centrifugal device. When compared to other buffer exchange methodologies, centrifugal diafiltration offered superior performance as measured by four key parameters (process time, desalting, recovery, integrity). Originally designed for resin-based affinity purification, the device also provides a platform for initial antibody purification or albumin carrier removal. Overall, the method offers a simplified workflow with reduced processing time and fewer hands-on requirements, without sacrificing labeling efficiency, final yield, or conjugate performance.