Disentangling epitope spacing and antibody flexibility from avidity: A quantitative model for competition and...

Multivalent binding of antibodies is boosted by avidity, a phenomenon that manifests as an increase in binding strength when an interaction occurs via several binding sites. Avidity in antibodies is typically measured using biosensor experiments such as surface plasmon resonance (SPR). However, the key step in formation of the closed, bivalent complex is optically silent, and currently no general methods can deconvolute the microscopic equilibrium constants that govern avidity. Here, we study antibody avidity binding to nanocalipers based on protein origami that place epitopes at well-controlled distances from 8 to 22 nm apart. We describe a new, improved bivalent model that accounts for the competition between bivalent 1:1 and 2:1 (antibody:antigen) complexes, thereby generating a comprehensive, robust, and fast model algorithm to fit avidity in SPR measurements. We use this to map the distance-dependent avidity enhancement of antibodies sampling a range of flexibilities and describe the underlying microstates populated during bivalent binding. With the improved model, we studied avidity at different epitope spacings and found a rather flat distance dependence in avidity for different epitope spacings. Our findings will improve the current workflow of quantifying avidity with SPR and can serve as a tool to achieve a more detailed insight into the mechanistic basis of bivalent binding in antibodies.