A case study on Sema3E-Fc aggregation and assay-dependent differences in quantitation
Related Experiment Video
Updated: May 23, 2026
Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses
Published on: April 1, 2022
Daniela Bumbaca1, Raymond K Tong, Alexander W Koch
1Department of Pharmacokinetic & Pharmacodynamic Sciences, Genentech, Inc., CA, USA.
Background:
In evaluating the serum concentrations in mice of a Sema3E IgG1 Fc fusion protein, a possible antitumor agent, two ELISAs were developed: a generic assay detecting only the Fc portion of the therapeutic and a specific receptor-binding assay detecting intact protein.
Results:
An unexpected discrepancy was observed in the measured in vivo serum concentrations, with the generic ELISA yielding higher concentrations than the specific ELISA. Size-exclusion HPLC and SDS-PAGE analysis of in vitro serum stability samples revealed extensive aggregation of Sema3E-Fc. The generic assay recovered more Sema3E-Fc in the presence of aggregates than the specific assay.
Conclusion:
Biophysical characterization combined with immunochemical analysis was key to elucidating not only the nature of the protein instability, but also the cause for the assay discrepancy.
