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

Abstract

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.

Dye-doped silica nanoparticles: synthesis, surface chemistry and bioapplications

“…Overall, while many reports describe the functionalisation of silica NPs with biomolecules as facile, it is in fact non-trivial and researchers need to carefully consider the bioconjugation chemistries used for attachment as it can affect biomolecule orientation on the NP surface (Saha et al 2014;Jeong et al 2017) and overall colloidal stability of functionalised NPs (Moore et al 2015). It is therefore appropriate to evaluate bioconjugation strategies based on the biological performance of the resultant biofunctionalised NP.…”

Effects of different quantities of antibody conjugated with magnetic nanoparticles on cell separation efficiency

“…Amide bond formation involves the use of EDC/NHS as a cross-linking agent to provide coupling with MNPs bearing carboxylic groups on their surface. Despite high efficiency, because these amine groups are mostly located within the antigen binding sites, the main potential drawbacks are occupation of antigen-binding sites [46] and low conjugation rate of Abs through amide bonds (24-27%) [43]. It was previously shown that when carbodiimide was used to functionalize carboxyl groups in MNPs to bind to amine groups in Abs, the availability of antibody binding site was less than 10% [44].…”

Advances in targeted nanotherapeutics: From bioconjugation to biomimicry

“…Further, for magnetic NPs such as iron oxide (which are particularly useful for contrast enhancement in dual imaging and therapeutic strategies), antibodies can be attached through an amidation reaction that uses EDC chemistry similar to that used for polymer particles [13]. Importantly, directionality of the antibody may play a role in its binding affinity, as the antigen-binding sites need to be accessible for efficient targeting [14, 15]. With the conjugation methods described previously, antibodies are randomly oriented, which reduces selective binding efficiency, may have a low coupling efficiency, and can result in NP aggregation [16].…”

“…With the conjugation methods described previously, antibodies are randomly oriented, which reduces selective binding efficiency, may have a low coupling efficiency, and can result in NP aggregation [16]. With directional conjugation methods, antibodies are directly linked to the NP by the Fc region of the antibody, leaving the antigen-binding region unhindered [14–17]. With these strategies, fewer antibodies are needed per NP to achieve the same targeting effect, thus reducing cost and enhancing performance [15, 17].…”