Highly robust and optimized conjugation of antibodies to nanoparticles using quantitatively validated protocols

Antibody-conjugated nanoparticles (NPs) have attracted great attention in diagnostic and therapeutic applications due to their high sensitivity and specificity for biotargets, as well as their wide applicability. Unfortunately, these features are significantly affected by antibody conjugation methods in terms of conjugation efficiency, orientation of the target binding site in the antibody, and denaturation during chemical conjugation reactions. Furthermore, the number of conjugated antibodies on each NP and the overall targeting efficacy are critical factors for a quantitative bioassay with antibody-conjugated NPs. Herein, we report a versatile and oriented antibody conjugation method using copper-free click chemistry. Moreover, the number of conjugated antibodies and their binding capacity were quantitatively and experimentally evaluated using fluorescently-labeled antibodies and antigens. The strong binding capability of antibody-conjugated NPs prepared using the copper-free click chemistry-based conjugation strategy was 8 times superior to the binding capability seen following the use of the EDC/NHS-coupling method. Additionally, the versatility of the developed antibody conjugation method was also demonstrated by conjugation of the antibody to three kinds of silica-encapsulated NPs.