Hybrid Supercharged Antibodies: A Rational Approach to Boost Immunoassay Sensitivity via Controlled Nanoparticle Adsorption.

This study presents a novel approach to enhance the sensitivity of a lateral flow immunoassay by computationally designing supercharged antibodies that optimize both the adsorption amount and molecular orientation on nanoparticle surfaces. We engineered immunoglobulin G antibodies with positively charged Fc domains and negatively charged Fab domains to create charge-polarized molecules for controlled interaction with negatively charged cellulose nanoparticles (NanoAct). The supercharged antibodies retained physicochemical properties and antigen-binding affinities identical to those of the wild-type antibody. Quantitative analysis showed that positively supercharged Fc domains enhanced antibody adsorption, and the charge-polarized design (featuring a negatively charged Fab and a positively charged Fc; c-10/Fc-pos14) enhanced relative Fab accessibility on the nanoparticle surface. Interaction analyses between supercharged antibodies and NanoAct using isothermal titration calorimetry quantitatively revealed that the c-10/Fc-pos14 antibody adsorbed onto NanoAct in the tail-on orientation. Consequently, lateral flow immunoassay performance tests demonstrated an 8-fold improvement in the limit of detection from 25 to 3.13 ng/mL, without increasing nonspecific binding. The key design principle involves maintaining sufficient charge separation between the domains to ensure proper orientation control. This supercharging approach represents a promising strategy for boosting immunoassay sensitivity while preserving sufficiently low noise levels with potential applications in other antibody-based diagnostic platforms.