Antibody conjugated nanoparticle probes allow for the study and visualization of cellular and molecular processes in vitro and in vivo; a critical tool for both basic science researchers and clinicians.
Many investigators spend excessive time and energy synthesizing these conjugates themselves with varying degrees of success and inconsistent results. Most find that while performing conjugations on their own, they suffer from inconsistent antibody-to-particle attachment and non-reproducible cell labeling efficiency.
Solving the first problem of inconsistent antibody-to-particle attachment requires both:
(1) creation of a stable linkage between the particle and the antibody and
(2) Proper characterization of the antibody attachment.
To create a stable linkage, antibodies should be covalently linked to the surface. The scheme below shows several different methods of antibody conjugation.
The physical adsorption method is the least stable. It relies on the affinity of certain functional groups on proteins such as amines, thiols, etc… to be attracted via non-covalent interactions to the particle surface. In an in vivo environment, or even in cell media, antibodies that are physically adsorbed can be easily displaced.
Thus, the covalent conjugation method is the most widely used regime. It provides a stable linkage between the particle and the antibody and several different chemistries such as N-Hydroxysuccinimide (NHS) coupled with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) can be employed. However, while this method produces a stable attachment, it still must be characterized.
DIRECTIONAL CONJUGATION FOR BETTER EFFICIENCY
Characterization usually involves estimating the antibody to particle ratio by comparing fluorescence measurements of fluorophore-labeled antibody conjugated particles to known fluorophore-labeled antibody standards. The measurement itself can be complicated by the fluorophore quenching at the metal nanoparticle surface. Furthermore, researchers also must prepare the fluorophore-conjugated antibody and its standards.
NanoHybrids employs a directional conjugation method which alleviates many of these problems.
To enable directional attachment, one end of a heterobifunctional crosslinker molecule is bound to the Fc region of the antibody. The other end of the linker binds directly to the nanoparticle surface, providing a directional linkage between the antibody and the particle. Using this method, a stable linkage between the particle and the antibody is realized while leaving the variable region (Fv), or antigen interacting site, sterically unhindered and available for binding.
The directional nature of the attachment makes characterization of the antibody-to-particle ratio more consistent and also enhances cell labeling efficiency. Labeling efficiency can be increased by 7 to 10 fold using a directional versus non-directional attachment method. Using NanoHybrids conjugated particles can yield significant cost savings, enhance productivity, and accelerate scientific discovery.
Our functionalized and/or antibody conjugated gold nanoparticles will be available soon. Please contact us if you have any questions about your antibody conjugation requirements.
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