Related Experiment Video
Updated: Jan 30, 2026

Determining Binding Affinity KD of Radiolabeled Antibodies to Immobilized Antigens
Published on: June 23, 2022
Optimization of Enzymatic Antibody Fragmentation for Yield, Efficiency, and Binding Affinity
Andrew W L Kinman1, Rebecca R Pompano1,2
1Department of Chemistry , University of Virginia , P.O. Box 400319, Charlottesville , Virginia 22904 , United States.
Abstract:
Enzymatic antibody fragmentation has been well studied for various hosts and isotypes, but fragmentation patterns also vary unpredictably by clone, and optimizing Fab or F(ab')2 production by trial and error consumes large quantities of antibodies. Here, we report a systematic strategy for optimizing functional F(ab')2 production via pepsin digestion from small quantities of IgG. We tested three key parameters that affect fragmentation, pH, enzyme concentration (% pepsin w/w), and reaction time, and found that pH had the greatest impact on fragmentation yield and efficiency. We then developed a systematic approach to obtaining acceptable yields, digestion efficiency, and binding affinity. Three case studies are described to illustrate the approach. We anticipate that this work will provide a quick and cost-effective method for researchers to produce antibody fragments from whole IgG, avoiding haphazard trial and error.
Related Concept Videos
Affinity and Avidity
ATP Yield
The ETC is embedded in the inner mitochondrial membrane and is comprised of four main protein complexes and an ATP synthase. NADH and FADH2 pass electrons to these complexes, which pump protons into the intermembrane space. This distribution of...
Reaction Yield
The Equilibrium Binding Constant and Binding Strength
Electron Affinity
Habitat Fragmentation

