Covalent binding of antibodies to the surface of gold nanoparticles results in robust and reliable conjugates. There are a number of advantages to covalent binding including:
- Less antibody is needed to maximize sensitivity, which reduces the overall cost of an assay.
- High stability can be obtained with covalent conjugates, which solves challenges associated with difficult sample matrices.
- Conjugates are easily prepared without requiring salt or pH optimizations, which saves time when performing antibody screening experiments.
- The antibody-to-particle ratio can be precisely controlled, which is important for adjusting the dynamic range in competitive assays and optimizing sensitivity with antibodies with varying binding kinetics.
The BioReady™ line of products from nanoComposix are optimized for use in conjugations and lateral flow diagnostic assays. Carboxyl functionalized nanoparticles are recommended for covalent binding. For this surface, EDC and sulfo-NHS reactive groups are used for amide bond formation, linking the carboxylic acid on the nanoparticle to primary amines in the lysine residues of the antibody or protein. A typical IgG antibody will have 80–100 lysine residues of which 30–40 will be accessible for EDC/NHS binding.
It is important to understand that each antibody is unique and may exhibit different performance when conjugated under different conditions compared to other proteins. There is no one-size-fits all conjugation method. In this module we outline a series of experiments designed to quickly and effectively produce robust and reliable covalent conjugates. If these instructions are carefully followed you will have a high chance of success. Building great conjugates is not easy, but once it works, they will perform again and again.